It is fairly common to have multiple read groups for a sample, either from sequencing multiple libraries or from spreading a library across multiple lanes. It seems this causes a lot of confusion, and people often tell us they're not sure how to organize the data for the pre-processing steps or how to feed the data into HaplotypeCaller.
Well, there are several options for organizing the processing. We have a fairly detailed FAQ article that describes our preferred workflow for pre-processing data from multiplexed sequencing and multi-library designs. But in this article we describe at a simpler level what are the main two options depending on how you want to provide the analysis ready BAM files to the variant caller.
The simplest thing to do is to input all the bam files that belong to that sample, either at the MarkDuplicates step, the Indel Realignment step or at the BQSR step. The choice depends mostly on how deep the coverage is. High depth means a lot of data to process at the same time, which slows down Indel Realignment. This is because Indel Realignment ignores all read group information and simply processes all reads together. BQSR doesn't suffer from that problem because it processes read groups separately. In either case, when you input all samples together, the bam that gets written out with the processed data will include all the libraries / read groups in one handy per-sample file.
Note: We do not require the PU field in the RG, however, BQSR will consider the PU field over all other fields.
Another option is to keep all the bam files separate until variant calling, and then input them to Haplotype Caller together. You can do this by simply running Indel Realignment and BQSR on each of the bams separately. You can then input all of the bams into HaplotypeCaller at once. This works even if you want to run HaplotypeCaller in GVCF mode, which can only be done on a single sample at a time. As long as the SM tags are identical, HaplotypeCaller will recognize that it's a single-sample run. This is because the GATK engine will merge the data before presenting it to the HaplotypeCaller tool, so HaplotypeCaller does not know nor care whether the data came from many files or one file.
Note: If you input many bam files into Indel Realigner, the default output is one bam file. However, you can output one bam file for each input bam file by using
We have not yet validated the joint genotyping methods (HaplotypeCaller in
-ERC GVCF mode per-sample then GenotypeGVCFs per-cohort) on RNAseq data. Our standard recommendation is to process RNAseq samples individually as laid out in the RNAseq-specific documentation.
However, we know that a lot of people have been trying out the joint genotyping workflow on RNAseq data, and there do not seem to be any major technical problems. You are welcome to try it on your own data, with the caveat that we cannot guarantee correctness of results, and may not be able to help you if something goes wrong. Please be sure to examine your results carefully and critically.
If you do pursue this, you will need to pre-process your samples according to our RNA-specific documentation, then switch to the GVCF workflow at the HaplotypeCaller stage. For filtering, it will be up to you to determine whether the hard filtering or VQSR filtering method produce best results. We have not tested any of this so we cannot provide a recommendation. Be prepared to do a lot of analysis to validate the quality of your results.
In general most GATK tools don't care about ploidy. The major exception is, of course, at the variant calling step: the variant callers need to know what ploidy is assumed for a given sample in order to perform the appropriate calculations.
As of version 3.3, the HaplotypeCaller and GenotypeGVCFs are able to deal with non-diploid organisms (whether haploid or exotically polyploid). In the case of HaplotypeCaller, you need to specify the ploidy of your non-diploid sample with the
-ploidy argument. HC can only deal with one ploidy at a time, so if you want to process different chromosomes with different ploidies (e.g. to call X and Y in males) you need to run them separately. On the bright side, you can combine the resulting files afterward. In particular, if you’re running the -ERC GVCF workflow, you’ll find that both CombineGVCFs and GenotypeGVCFs are able to handle mixed ploidies (between locations and between samples). Both tools are able to correctly work out the ploidy of any given sample at a given site based on the composition of the GT field, so they don’t require you to specify the -ploidy argument.
For earlier versions (all the way to 2.0) the fallback option is UnifiedGenotyper, which also accepts the
For normal organism ploidy, you just set the
-ploidy argument to the desired number of chromosomes per organism. In the case of pooled sequencing experiments, this argument should be set to the number of chromosomes per barcoded sample, i.e.
(Ploidy per individual) * (Individuals in pool).
Several variant annotations are not appropriate for use with non-diploid cases. In particular, InbreedingCoeff will not be annotated on non-diploid calls. Annotations that do work and are supported in non-diploid use cases are the following:
AF, and Genotype annotations such as
You should also be aware of the fundamental accuracy limitations of high ploidy calling. Calling low-frequency variants in a pool or in an organism with high ploidy is hard because these rare variants become almost indistinguishable from sequencing errors.
Short answer: NO.
Medium answer: no, at least not if you want to run a low-risk pipeline.
Long answer: see below for details.
There are several reasons why you might want to do this: you're using the latest version of GATK and one of the tools has a show-stopping bug, so you'd like to use an older, pre-bug version of that tool, but still use the latest version of all the other tools; or maybe you've been using an older version of GATK and you'd like to use a new tool, but keep using the rest in the version that you've been using to process hundreds of samples already.
The problem: compatibility is not guaranteed
In many cases, when we modify one tool in the GATK, we need to make adjustments to other tools that interact either directly or indirectly with the data consumed or produced by the upgraded tool. If you mix and match tools from different versions of GATK, you risk running into compatibility issues. For example, HaplotypeCaller expects a BAM compressed by Reduce Reads to have its data annotated in a certain way. If the information is formatted differently than what the HC expects (because that's how the corresponding RR from the same version does it), it can blow up -- or worse, do the wrong thing but not tell you there's a problem.
But what if the tools/tasks are in unrelated workflows?
Would it really be so bad to use CountReads from GATK version 2.7 for a quick QC check that's not actually part of my pipeline, which uses version 2.5? Well, maaaaybe not, but we still think it's a source of error, and we do our damnedest to eliminate those.
You shouldn't use tools from different versions within the same workflow, that's for sure. We don't think it's worth the risks. If there's a show-stopping bug, let us know and we promise to fix it as soon as (humanly) possible. For the rest, either accept that you're stuck with the version you started your study with (we may be able to help with workarounds for known issues), or upgrade your entire workflow and start your analysis from scratch. Depending on how far along you are one of those options will be less painful to you; go with that.
The plea bargain, and a warning
If despite our dire warnings you're still going to mix and match tool versions, fine, we can't stop you. But be really careful, and check every version release notes document ever. And keep in mind that when things go wrong, we will deny you support if we think you've been reckless.
We support the Variant Call Format (VCF) for variant callsets. No other file formats are supported.
No, we like VCF and we think it's important to have a good standard format. Multiplying formats just makes life hard for everyone, both developers and analysts.
The GATK supports the BAM format for reads, quality scores, alignments, and metadata (e.g. the lane of sequencing, center of origin, sample name, etc.). Starting with version 3.5, the CRAM format is supported as well. SAM format is not supported but can be easily converted with Picard tools.
The GATK doesn't have any tools for getting data into BAM format, but many other toolkits exist for this purpose. We recommend you look at Picard and Samtools for creating and manipulating BAM files. Also, many aligners are starting to emit BAM files directly. See BWA for one such aligner.
All BAM/CRAM files must satisfy the following requirements:
See the official BAM specification for more information on what constitutes a valid BAM file.
It depends on whether you're using the NCBI/GRC build 36/build 37 version of the human genome, or the UCSC hg18/hg19 version of the human genome. While substantially equivalent, the naming conventions are different. The canonical ordering of contigs for these genomes is as follows:
Human genome reference consortium standard ordering and names (b3x): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, MT...
UCSC convention (hg1x): chrM, chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY...
The easiest way to do it is to download Samtools and run the following command to examine the header of your file:
$ samtools view -H /path/to/my.bam @HD VN:1.0 GO:none SO:coordinate @SQ SN:1 LN:247249719 @SQ SN:2 LN:242951149 @SQ SN:3 LN:199501827 @SQ SN:4 LN:191273063 @SQ SN:5 LN:180857866 @SQ SN:6 LN:170899992 @SQ SN:7 LN:158821424 @SQ SN:8 LN:146274826 @SQ SN:9 LN:140273252 @SQ SN:10 LN:135374737 @SQ SN:11 LN:134452384 @SQ SN:12 LN:132349534 @SQ SN:13 LN:114142980 @SQ SN:14 LN:106368585 @SQ SN:15 LN:100338915 @SQ SN:16 LN:88827254 @SQ SN:17 LN:78774742 @SQ SN:18 LN:76117153 @SQ SN:19 LN:63811651 @SQ SN:20 LN:62435964 @SQ SN:21 LN:46944323 @SQ SN:22 LN:49691432 @SQ SN:X LN:154913754 @SQ SN:Y LN:57772954 @SQ SN:MT LN:16571 @SQ SN:NT_113887 LN:3994 ...
If the order of the contigs here matches the contig ordering specified above, and the
SO:coordinate flag appears in your header, then your contig and read ordering satisfies the GATK requirements.
Picard offers a tool called SortSam that will sort a BAM file properly. A similar utility exists in Samtools, but we recommend the Picard tool because SortSam will also set a flag in the header that specifies that the file is correctly sorted, and this flag is necessary for the GATK to know it is safe to process the data. Also, you can use the ReorderSam command to make a BAM file SQ order match another reference sequence.
A quick Unix command using Samtools will do the trick:
$ samtools view -H /path/to/my.bam | grep '^@RG' @RG ID:0 PL:solid PU:Solid0044_20080829_1_Pilot1_Ceph_12414_B_lib_1_2Kb_MP_Pilot1_Ceph_12414_B_lib_1_2Kb_MP LB:Lib1 PI:2750 DT:2008-08-28T20:00:00-0400 SM:NA12414 CN:bcm @RG ID:1 PL:solid PU:0083_BCM_20080719_1_Pilot1_Ceph_12414_B_lib_1_2Kb_MP_Pilot1_Ceph_12414_B_lib_1_2Kb_MP LB:Lib1 PI:2750 DT:2008-07-18T20:00:00-0400 SM:NA12414 CN:bcm @RG ID:2 PL:LS454 PU:R_2008_10_02_06_06_12_FLX01080312_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC @RG ID:3 PL:LS454 PU:R_2008_10_02_06_07_08_rig19_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC @RG ID:4 PL:LS454 PU:R_2008_10_02_17_50_32_FLX03080339_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC ...
The presence of the
@RG tags indicate the presence of read groups. Each read group has a
SM tag, indicating the sample from which the reads belonging to that read group originate.
In addition to the presence of a read group in the header, each read must belong to one and only one read group. Given the following example reads,
$ samtools view /path/to/my.bam | grep '^@RG' EAS139_44:2:61:681:18781 35 1 1 0 51M = 9 59 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA B<>;==?=?<==?=?=>>?>><=<?=?8<=?>?<:=?>?<==?=>:;<?:= RG:Z:4 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 EAS139_44:7:84:1300:7601 35 1 1 0 51M = 12 62 TAACCCTAAGCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA G<>;==?=?&=>?=?<==?>?<>>?=?<==?>?<==?>?1==@>?;<=><; RG:Z:3 MF:i:18 Aq:i:0 NM:i:1 UQ:i:5 H0:i:0 H1:i:85 EAS139_44:8:59:118:13881 35 1 1 0 51M = 2 52 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA @<>;<=?=?==>?>?<==?=><=>?-?;=>?:><==?7?;<>?5?<<=>:; RG:Z:1 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 EAS139_46:3:75:1326:2391 35 1 1 0 51M = 12 62 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA @<>==>?>@???B>A>?>A?A>??A?@>?@A?@;??A>@7>?>>@:>=@;@ RG:Z:0 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 ...
membership in a read group is specified by the
RG:Z:* tag. For instance, the first read belongs to read group 4 (sample NA11881), while the last read shown here belongs to read group 0 (sample NA12414).
Yes! Many algorithms in the GATK need to know that certain reads were sequenced together on a specific lane, as they attempt to compensate for variability from one sequencing run to the next. Others need to know that the data represents not just one, but many samples. Without the read group and sample information, the GATK has no way of determining this critical information. You can use Picard's AddOrReplaceReadGroups tool to add read group information.
You can use the GATK to do the following:
java -jar GenomeAnalysisTK.jar -R reference.fasta -I full_input.bam -T PrintReads -L chr1:10-20 -o subset_input.bam
and you'll get a BAM file containing only reads overlapping those points. This operation retains the complete BAM header from the full file (this was the reference aligned to, after all) so that the BAM remains easy to work with. We routinely use these features for testing and high-performance analysis with the GATK.
Absolutely. Just use the
-L argument to provide the list of intervals you wish to run on. Or you can use
-XL to exclude intervals, e.g. to blacklist genome regions that are problematic.
GATK supports several types of interval list formats: Picard-style
.list, BED files with extension
.bed, and VCF files.
Picard-style interval files have a SAM-like header that includes a sequence dictionary. The intervals are given in the form
<chr> <start> <stop> + <target_name>, with fields separated by tabs, and the coordinates are 1-based (first position in the genome is position 1, not position 0).
@HD VN:1.0 SO:coordinate @SQ SN:1 LN:249250621 AS:GRCh37 UR:http://www.broadinstitute.org/ftp/pub/seq/references/Homo_sapiens_assembly19.fasta M5:1b22b98cdeb4a9304cb5d48026a85128 SP:Homo Sapiens @SQ SN:2 LN:243199373 AS:GRCh37 UR:http://www.broadinstitute.org/ftp/pub/seq/references/Homo_sapiens_assembly19.fasta M5:a0d9851da00400dec1098a9255ac712e SP:Homo Sapiens 1 30366 30503 + target_1 1 69089 70010 + target_2 1 367657 368599 + target_3 1 621094 622036 + target_4 1 861320 861395 + target_5 1 865533 865718 + target_6
This is the preferred format because the explicit sequence dictionary safeguards against accidental misuse (e.g. apply hg18 intervals to an hg19 BAM file). Note that this file is 1-based, not 0-based (the first position in the genome is position 1).
This is a simpler format, where intervals are in the form
<chr>:<start>-<stop>, and no sequence dictionary is necessary. This file format also uses 1-based coordinates. Note that only the
<chr> part is strictly required; if you just want to specify chromosomes/ contigs as opposed to specific coordinate ranges, you don't need to specify the rest. Both
<chr> can be present in the same file. You can also specify intervals in this format directly at the command line instead of writing them in a file.
We also accept the widely-used BED format, where intervals are in the form
<chr> <start> <stop>, with fields separated by tabs. However, you should be aware that this file format is 0-based for the start coordinates, so coordinates taken from 1-based formats (e.g. if you're cooking up a custom interval list derived from a file in a 1-based format) should be offset by 1. The GATK engine recognizes the
.bed extension and interprets the coordinate system accordingly.
Yeah, I bet you didn't expect that was a thing! It's very convenient. Say you want to redo a variant calling run on a set of variant calls that you were given by a colleague, but with the latest version of HaplotypeCaller. You just provide the VCF, slap on some padding on the fly using e.g.
-ip 100 in the HC command, and boom, done. Each record in the VCF will be interpreted as a single-base interval, and by adding padding you ensure that the caller sees enough context to reevaluate the call appropriately.
Yes, thanks for asking. The intervals MUST be sorted by coordinate (in increasing order) within contigs; and the contigs must be sorted in the same order as in the sequence dictionary. This is for efficiency reasons.
Sure, no problem -- just pass them in using separate
-L arguments. You can use all the different formats within the same command line. By default, the GATK engine will take the UNION of all the intervals in all the sets. This behavior can be modified by setting an
Very gracefully. By default the GATK engine will merge any intervals that abut (i.e. they are contiguous, they touch without overlapping) or overlap into a single interval. This behavior can be modified by setting an
You can use the
-ip engine argument to add padding on the fly. No need to produce separate padded targets files. Sweet, right?
Note that if intervals that previously didn't abut or overlap before you added padding now do so, by default the GATK engine will merge them as described above. This behavior can be modified by setting an
NOTE: This article will be deprecated in the near future as this information will be consolidated elsewhere.
We make various files available for public download from the GSA FTP server, such as the GATK resource bundle and presentation slides. We also maintain a public upload feature for processing bug reports from users.
There are two logins to choose from depending on whether you want to upload or download something:
location: ftp.broadinstitute.org username: gsapubftp-anonymous password: <blank>
location: ftp.broadinstitute.org username: gsapubftp password: 5WvQWSfi
If you use your browser as FTP client, make sure to include the login information in the address, otherwise you will access the general Broad Institute FTP instead of our team FTP. This should work as a direct link (for downloading only):
Most GATK tools apply several read filters by default. You can look up exactly what are the defaults for each tool in their respective Technical Documentation pages.
But sometimes you want to specify additional filters yourself (and before you ask, no, you cannot disable the default read filters used by a given tool). This is how you do it:
--read-filter argument (or
-rf for short) allows you to apply whatever read filters you'd like. For example, to add the
MaxReadLengthFilter filter above to
PrintReads, you just add this to your command line:
The read filter will be applied with its default value (which you can also look up in the Tech Docs for that filter). Now, if you want to specify a different value from the default, you pass the relevant argument by adding this right after the read filter:
--read_filter MaxReadLength -maxReadLength 76
It's important that you pass the argument right after the filter itself, otherwise the command line parser won't know that they're supposed to go together.
And of course, you can add as many filters as you like by using multiple copies of the
--read_filter MaxReadLength --maxReadLength 76 --read_filter ZeroMappingQualityRead
This article describes the steps necessary to prepare your reference file (if it's not one that you got from us). As a complement to this article, see the relevant tutorial.
The GATK uses two files to access and safety check access to the reference files: a
.dict dictionary of the contig names and sizes and a
.fai fasta index file to allow efficient random access to the reference bases. You have to generate these files in order to be able to use a Fasta file as reference.
NOTE: Picard and samtools treat spaces in contig names differently. We recommend that you avoid using spaces in contig names.
We use CreateSequenceDictionary.jar from Picard to create a .dict file from a fasta file.
> java -jar CreateSequenceDictionary.jar R= Homo_sapiens_assembly18.fasta O= Homo_sapiens_assembly18.dict [Fri Jun 19 14:09:11 EDT 2009] net.sf.picard.sam.CreateSequenceDictionary R= Homo_sapiens_assembly18.fasta O= Homo_sapiens_assembly18.dict [Fri Jun 19 14:09:58 EDT 2009] net.sf.picard.sam.CreateSequenceDictionary done. Runtime.totalMemory()=2112487424 44.922u 2.308s 0:47.09 100.2% 0+0k 0+0io 2pf+0w
This produces a SAM-style header file describing the contents of our fasta file.
> cat Homo_sapiens_assembly18.dict @HD VN:1.0 SO:unsorted @SQ SN:chrM LN:16571 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:d2ed829b8a1628d16cbeee88e88e39eb @SQ SN:chr1 LN:247249719 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:9ebc6df9496613f373e73396d5b3b6b6 @SQ SN:chr2 LN:242951149 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:b12c7373e3882120332983be99aeb18d @SQ SN:chr3 LN:199501827 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:0e48ed7f305877f66e6fd4addbae2b9a @SQ SN:chr4 LN:191273063 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:cf37020337904229dca8401907b626c2 @SQ SN:chr5 LN:180857866 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:031c851664e31b2c17337fd6f9004858 @SQ SN:chr6 LN:170899992 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:bfe8005c536131276d448ead33f1b583 @SQ SN:chr7 LN:158821424 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:74239c5ceee3b28f0038123d958114cb @SQ SN:chr8 LN:146274826 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:1eb00fe1ce26ce6701d2cd75c35b5ccb @SQ SN:chr9 LN:140273252 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:ea244473e525dde0393d353ef94f974b @SQ SN:chr10 LN:135374737 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:4ca41bf2d7d33578d2cd7ee9411e1533 @SQ SN:chr11 LN:134452384 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:425ba5eb6c95b60bafbf2874493a56c3 @SQ SN:chr12 LN:132349534 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:d17d70060c56b4578fa570117bf19716 @SQ SN:chr13 LN:114142980 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:c4f3084a20380a373bbbdb9ae30da587 @SQ SN:chr14 LN:106368585 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:c1ff5d44683831e9c7c1db23f93fbb45 @SQ SN:chr15 LN:100338915 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:5cd9622c459fe0a276b27f6ac06116d8 @SQ SN:chr16 LN:88827254 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:3e81884229e8dc6b7f258169ec8da246 @SQ SN:chr17 LN:78774742 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:2a5c95ed99c5298bb107f313c7044588 @SQ SN:chr18 LN:76117153 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:3d11df432bcdc1407835d5ef2ce62634 @SQ SN:chr19 LN:63811651 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:2f1a59077cfad51df907ac25723bff28 @SQ SN:chr20 LN:62435964 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:f126cdf8a6e0c7f379d618ff66beb2da @SQ SN:chr21 LN:46944323 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:f1b74b7f9f4cdbaeb6832ee86cb426c6 @SQ SN:chr22 LN:49691432 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:2041e6a0c914b48dd537922cca63acb8 @SQ SN:chrX LN:154913754 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:d7e626c80ad172a4d7c95aadb94d9040 @SQ SN:chrY LN:57772954 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:62f69d0e82a12af74bad85e2e4a8bd91 @SQ SN:chr1_random LN:1663265 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:cc05cb1554258add2eb62e88c0746394 @SQ SN:chr2_random LN:185571 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:18ceab9e4667a25c8a1f67869a4356ea @SQ SN:chr3_random LN:749256 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:9cc571e918ac18afa0b2053262cadab6 @SQ SN:chr4_random LN:842648 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:9cab2949ccf26ee0f69a875412c93740 @SQ SN:chr5_random LN:143687 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:05926bdbff978d4a0906862eb3f773d0 @SQ SN:chr6_random LN:1875562 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:d62eb2919ba7b9c1d382c011c5218094 @SQ SN:chr7_random LN:549659 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:28ebfb89c858edbc4d71ff3f83d52231 @SQ SN:chr8_random LN:943810 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:0ed5b088d843d6f6e6b181465b9e82ed @SQ SN:chr9_random LN:1146434 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:1e3d2d2f141f0550fa28a8d0ed3fd1cf @SQ SN:chr10_random LN:113275 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:50be2d2c6720dabeff497ffb53189daa @SQ SN:chr11_random LN:215294 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:bfc93adc30c621d5c83eee3f0d841624 @SQ SN:chr13_random LN:186858 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:563531689f3dbd691331fd6c5730a88b @SQ SN:chr15_random LN:784346 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:bf885e99940d2d439d83eba791804a48 @SQ SN:chr16_random LN:105485 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:dd06ea813a80b59d9c626b31faf6ae7f @SQ SN:chr17_random LN:2617613 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:34d5e2005dffdfaaced1d34f60ed8fc2 @SQ SN:chr18_random LN:4262 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:f3814841f1939d3ca19072d9e89f3fd7 @SQ SN:chr19_random LN:301858 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:420ce95da035386cc8c63094288c49e2 @SQ SN:chr21_random LN:1679693 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:a7252115bfe5bb5525f34d039eecd096 @SQ SN:chr22_random LN:257318 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:4f2d259b82f7647d3b668063cf18378b @SQ SN:chrX_random LN:1719168 UR:file:/humgen/gsa-scr1/depristo/dev/GenomeAnalysisTK/trunk/Homo_sapiens_assembly18.fasta M5:f4d71e0758986c15e5455bf3e14e5d6f
We use the faidx command in samtools to prepare the fasta index file. This file describes byte offsets in the fasta file for each contig, allowing us to compute exactly where a particular reference base at contig:pos is in the fasta file.
> samtools faidx Homo_sapiens_assembly18.fasta 108.446u 3.384s 2:44.61 67.9% 0+0k 0+0io 0pf+0w
This produces a text file with one record per line for each of the fasta contigs. Each record is of the: contig, size, location, basesPerLine, bytesPerLine. The index file produced above looks like:
> cat Homo_sapiens_assembly18.fasta.fai chrM 16571 6 50 51 chr1 247249719 16915 50 51 chr2 242951149 252211635 50 51 chr3 199501827 500021813 50 51 chr4 191273063 703513683 50 51 chr5 180857866 898612214 50 51 chr6 170899992 1083087244 50 51 chr7 158821424 1257405242 50 51 chr8 146274826 1419403101 50 51 chr9 140273252 1568603430 50 51 chr10 135374737 1711682155 50 51 chr11 134452384 1849764394 50 51 chr12 132349534 1986905833 50 51 chr13 114142980 2121902365 50 51 chr14 106368585 2238328212 50 51 chr15 100338915 2346824176 50 51 chr16 88827254 2449169877 50 51 chr17 78774742 2539773684 50 51 chr18 76117153 2620123928 50 51 chr19 63811651 2697763432 50 51 chr20 62435964 2762851324 50 51 chr21 46944323 2826536015 50 51 chr22 49691432 2874419232 50 51 chrX 154913754 2925104499 50 51 chrY 57772954 3083116535 50 51 chr1_random 1663265 3142044962 50 51 chr2_random 185571 3143741506 50 51 chr3_random 749256 3143930802 50 51 chr4_random 842648 3144695057 50 51 chr5_random 143687 3145554571 50 51 chr6_random 1875562 3145701145 50 51 chr7_random 549659 3147614232 50 51 chr8_random 943810 3148174898 50 51 chr9_random 1146434 3149137598 50 51 chr10_random 113275 3150306975 50 51 chr11_random 215294 3150422530 50 51 chr13_random 186858 3150642144 50 51 chr15_random 784346 3150832754 50 51 chr16_random 105485 3151632801 50 51 chr17_random 2617613 3151740410 50 51 chr18_random 4262 3154410390 50 51 chr19_random 301858 3154414752 50 51 chr21_random 1679693 3154722662 50 51 chr22_random 257318 3156435963 50 51 chrX_random 1719168 3156698441 50 51
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This document provides technical details and recommendations on how the parallelism options offered by the GATK can be used to yield optimal performance results.
There are two options for multi-threading with the GATK, controlled by the arguments
-nct, respectively, which can be combined:
-nt / --num_threadscontrols the number of data threads sent to the processor
-nct / --num_cpu_threads_per_data_threadcontrols the number of CPU threads allocated to each data thread
For more information on how these multi-threading options work, please read the primer on parallelism for the GATK.
Each data thread needs to be given the full amount of memory you’d normally give a single run. So if you’re running a tool that normally requires 2 Gb of memory to run, if you use
-nt 4, the multithreaded run will use 8 Gb of memory. In contrast, CPU threads will share the memory allocated to their “mother” data thread, so you don’t need to worry about allocating memory based on the number of CPU threads you use.
-nctwith versions 2.2 and 2.3
Because of the way the
-nct option was originally implemented, in versions 2.2 and 2.3, there is one CPU thread that is reserved by the system to “manage” the rest. So if you use
-nct, you’ll only really start seeing a speedup with
-nct 3 (which yields two effective "working" threads) and above. This limitation has been resolved in the implementation that will be available in versions 2.4 and up.
Please note that not all tools support all parallelization modes. The parallelization modes that are available for each tool depend partly on the type of traversal that the tool uses to walk through the data, and partly on the nature of the analyses it performs.
|Tool||Full name||Type of traversal||NT||NCT||SG|
Note that while HaplotypeCaller supports
-nct in principle, many have reported that it is not very stable (random crashes may occur -- but if there is no crash, results will be correct). We prefer not to use this option with HC; use it at your own risk.
The table below summarizes configurations that we typically use for our own projects (one per tool, except we give three alternate possibilities for the UnifiedGenotyper). The different values allocated for each tool reflect not only the technical capabilities of these tools (which options are supported), but also our empirical observations of what provides the best tradeoffs between performance gains and commitment of resources. Please note however that this is meant only as a guide, and that we cannot give you any guarantee that these configurations are the best for your own setup. You will probably have to experiment with the settings to find the configuration that is right for you.
|Cluster nodes||1||4||4||1||4||4||4 / 4 / 4|
|CPU threads (
||1||1||8||4-8||1||4||3 / 6 / 24|
|Data threads (
||24||1||1||1||1||1||8 / 4 / 1|
|Memory (Gb)||48||4||4||4||4||16||32 / 16 / 4|
Where NT is data multithreading, NCT is CPU multithreading and SG is scatter-gather using Queue or other data parallelization framework. For more details on scatter-gather, see the primer on parallelism for the GATK and the documentation on pipelining options.
Note: only do this if you have been explicitly asked to do so.
You posted a question about a problem you had with GATK tools, we answered that we think it's a bug, and we asked you to submit a detailed bug report.
A snippet file is a slice of the original BAM file which contains the problematic region and is sufficient to reproduce the error. We need it in order to reproduce the problem on our end, which is the first necessary step to finding and fixing the bug. We ask you to provide this as a snippet rather than the full file so that you don't have to upload (and we don't have to process) huge giga-scale files.
-Largument and progressively narrowing down the interval
-Lto write the problematic region (with 500 bp padding on either side) to a new file -- this is your snippet file.
Upload them onto our FTP server with the following credentials:
location: ftp.broadinstitute.org username: gsapubftp password: 5WvQWSfi
Imagine a simple question like, "What's the depth of coverage at position A of the genome?"
First, you are given billions of reads that are aligned to the genome but not ordered in any particular way (except perhaps in the order they were emitted by the sequencer). This simple question is then very difficult to answer efficiently, because the algorithm is forced to examine every single read in succession, since any one of them might span position A. The algorithm must now take several hours in order to compute this value.
Instead, imagine the billions of reads are now sorted in reference order (that is to say, on each chromosome, the reads are stored on disk in the same order they appear on the chromosome). Now, answering the question above is trivial, as the algorithm can jump to the desired location, examine only the reads that span the position, and return immediately after those reads (and only those reads) are inspected. The total number of reads that need to be interrogated is only a handful, rather than several billion, and the processing time is seconds, not hours.
This reference-ordered sorting enables the GATK to process terabytes of data quickly and without tremendous memory overhead. Most GATK tools run very quickly and with less than 2 gigabytes of RAM. Without this sorting, the GATK cannot operate correctly. Thus, it is a fundamental rule of working with the GATK, which is the reason for the Central Dogma of the GATK:
To date we have published three papers on GATK (citation details below). The ideal way to cite the GATK is to use all as a triple citation, as in:
We sequenced 10 samples on 10 lanes on an Illumina HiSeq 2000, aligned the resulting reads to the hg19 reference genome with BWA (Li & Durbin), applied GATK (McKenna et al., 2010) base quality score recalibration, indel realignment, duplicate removal, and performed SNP and INDEL discovery and genotyping across all 10 samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations (DePristo et al., 2011; Van der Auwera et al., 2013).
The first GATK paper covers the computational philosophy underlying the GATK and is a good citation for the GATK in general.
The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA, 2010 GENOME RESEARCH 20:1297-303
The second GATK paper describes in more detail some of the key tools commonly used in the GATK for high-throughput sequencing data processing and variant discovery. The paper covers base quality score recalibration, indel realignment, SNP calling with UnifiedGenotyper, variant quality score recalibration and their application to deep whole genome, whole exome, and low-pass multi-sample calling. This is a good citation if you use the GATK for variant discovery.
A framework for variation discovery and genotyping using next-generation DNA sequencing data DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M, 2011 NATURE GENETICS 43:491-498
Note that the workflow described in this paper corresponds to the version 1.x to 2.x best practices. Some key steps for variant discovery have been significantly modified in later versions (3.x onwards). This paper should not be used as a definitive guide to variant discovery with GATK. For that, please see our online documentation guide.
The third GATK paper describes the Best Practices for Variant Discovery (version 2.x). It is intended mainly as a learning resource for first-time users and as a protocol reference. This is a good citation to include in a Materials and Methods section.
From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline Van der Auwera GA, Carneiro M, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M, 2013 CURRENT PROTOCOLS IN BIOINFORMATICS 43:11.10.1-11.10.33
Remember that as our work continues and our Best Practices recommendations evolve, specific command lines, argument values and even tool choices described in the paper become obsolete. Be sure to always refer to our Best Practices documentation for the most up-to-date and version-appropriate recommendations.
Our Best Practices pre-processing documentation assumes a simple experimental design in which you have one set of input sequence files (forward/reverse or interleaved FASTQ, or unmapped uBAM) per sample, and you run each step of the pre-processing workflow separately for each sample, resulting in one BAM file per sample at the end of this phase.
However, if you are generating multiple libraries for each sample, and/or multiplexing samples within and/or across sequencing lanes, the data must be de-multiplexed before pre-processing, typically resulting in multiple sets of FASTQ files per sample all of which should have distinct read group IDs (RGID).
At that point there are several different valid strategies for implementing the pre-processing workflow. Here at the Broad Institute, we run the initial steps of the pre-processing workflow (mapping, sorting and marking duplicates) separately on each individual read group. Then we merge the data to produce a single BAM file for each sample (aggregation); this is done by re-running Mark Duplicates, this time on all read group BAM files for a sample at the same time. Then we run Indel Realignment and Base Recalibration on the aggregated per-sample BAM files. See the worked-out example below and this presentation for more details.
Note that there are many possible ways to achieve a similar result; here we present the way we think gives the best combination of efficiency and quality. This assumes that you are dealing with one or more samples, and each of them was sequenced on one or more lanes.
Let's say we have this example data (assuming interleaved FASTQs containing both forward and reverse reads) for two sample libraries, sampleA and sampleB, which were each sequenced on two lanes, lane1 and lane2:
These will each be identified as separate read groups A1, A2, B1 and B2. If we had multiple libraries per sample, we would further distinguish them (eg sampleA_lib1_lane1.fq leading to read group A11, sampleA_lib2_lane1.fq leading to read group A21 and so on).
Assuming that you received one FASTQ file per sample library, per lane of sequence data (which amounts to a read group), run each file through mapping and sorting. During the mapping step you assign read group information, which will be very important in the next steps so be sure to do it correctly. See the read groups dictionary entry for guidance.
The example data becomes:
At this point we mark duplicates in each read group BAM file (dedup), which allows us to estimate the complexity of the corresponding library of origin as a quality control step. This step is optional.
The example data becomes:
Technically this first run of marking duplicates is not necessary because we will run it again per-sample, and that per-sample marking would be enough to achieve the desired result. To reiterate, we only do this round of marking duplicates for QC purposes.
Once you have pre-processed each read group individually, you merge read groups belonging to the same sample into a single BAM file. You can do this as a standalone step, bur for the sake of efficiency we combine this with the per-readgroup duplicate marking step (it's simply a matter of passing the multiple inputs to MarkDuplicates in a single command).
The example data becomes:
To be clear, this is the round of marking duplicates that matters. It eliminates PCR duplicates (arising from library preparation) across all lanes in addition to optical duplicates (which are by definition only per-lane).
Then you run indel realignment (optional) and base recalibration (BQSR).
The example data becomes:
Realigning around indels per-sample leads to consistent alignments across all lanes within a sample. This step is only necessary if you will be using a locus-based variant caller like MuTect 1 or UnifiedGenotyper (for legacy reasons). If you will be using HaplotypeCaller or MuTect2, you do not need to perform indel realignment.
Base recalibration will be applied per-read group if you assigned appropriate read group information in your data. BaseRecalibrator distinguishes read groups by RGID, or RGPU if it is available (PU takes precedence over ID). This will identify separate read groups (distinguishing both lanes and libraries) as such even if they are in the same BAM file, and it will always process them separately -- as long as the read groups are identified correctly of course. There would be no sense in trying to recalibrate across lanes, since the purpose of this processing step is to compensate for the errors made by the machine during sequencing, and the lane is the base unit of the sequencing machine (assuming the equipment is Illumina HiSeq or similar technology).
People often ask also if it's worth the trouble to try realigning across all samples in a cohort. The answer is almost always no, unless you have very shallow coverage. The problem is that while it would be lovely to ensure consistent alignments around indels across all samples, the computational cost gets too ridiculous too fast. That being said, for contrastive calling projects -- such as cancer tumor/normals -- we do recommend realigning both the tumor and the normal together in general to avoid slight alignment differences between the two tissue types.
The Panel of Normals (PoN) plays two important roles in somatic variant analysis:
Given these roles, the most important selection criteria are the technical properties of how the normal data was generated. It's very important to use normals that are as technically similar as possible to the tumor. Also, the samples should come from subjects that were young and healthy (to minimize the chance of using as normal a sample from someone who has an undiagnosed tumor).
If possible it is better to use normals generated from the same type of tissue because if the tissues were preserved differently, the artifact patterns may be different.
If this is your first rodeo, you're probably asking yourself:
What can GATK do for me? Identify variants in a bunch of sample sequences, with great sensitivity and specificity.
No but really, how do I know what to do? For each step in the Best Practices, there is a tutorial that details how to run the tools involved, with example commands. The idea is to daisy-chain all thosee tutorials in the order that they're referenced in the Best Practices doc into a pipeline.
Oh, you mean I can just copy/paste all the tutorial commands as they are?
Not quite, because there are a few things that need to be tweaked. For example, the tutorials use the
-L/--intervals argument to restrict analysis for demo purposes, but depending on your data and experimental design, you may need to remove it (e.g. for WGS) or adapt it (for WEx). Hopefully it's explained clearly enough in the tutorials.
Why don't you just provide one script that runs all the tools? It's really hard to build and maintain a one-size-fits-all pipeline solution. Really really hard. And not nearly as much fun as developing new analysis methods. We do provide a pipelining program called Queue that has the advantage of understanding GATK argument syntax natively, but you still have to actually write scripts yourself in Scala to use it. Sorry. Maybe one day we will be able to offer GATK analysis on the Cloud. But not today.
What if I want to know what a command line argument does or change a parameter? First, check out the basic GATK command syntax FAQ if it's your first time using GATK, then consult the relevant Tool Documentation page. Keep in mind that some arguments are "engine parameters" that are shared by many tools, and are listed in a separate document. Also, you can always use the search box to find an argument description really quickly.
The documentation seems chaotic. Is there any logic to how it's organized? Sort of. (And, ouch. Tough crowd.) The main category names should be obvious enough (if not, see the "Documentation Categories" tab). Within categories, everything is just in alphabetical order. In future, we're going to try to provide more use-case based structure, but for now this is what we have. The best way to find practical information is to either go from the Best Practices doc (which provide links to all FAQs, method articles and tutorials directly related to a given step), or use the search box and search-by-tag functions (see the "Search tab"). Be sure to also check out the Presentations section, which provides workshop materials and videos that explain a lot of the motivation and methods behind the Best Practices.
Does GATK include other tools beside the ones in the Best Practices? Oh sure, there's a whole bunch of them, all listed in the Tool Documentation section, categorized by type of analysis. But be aware that anything that's not part of the Best Practices is most likely either a tool that was written for a one-off analysis years ago, an experimental feature that we're still not sure is actually useful, or an accessory utility that can be used in many different ways and takes expert inside knowledge to use properly. All these may be buggy, insufficiently documented, or both. We provide support for them as well as humanly possible but ultimately, you use them at your own risk.
Why do the answers to these questions keep getting longer and longer? I don't know what you're talking about.
There are four major organizational units for next-generation DNA sequencing processes that used throughout the GATK documentation:
Lane: The basic machine unit for sequencing. The lane reflects the basic independent run of an NGS machine. For Illumina machines, this is the physical sequencing lane.
Library: A unit of DNA preparation that at some point is physically pooled together. Multiple lanes can be run from aliquots from the same library. The DNA library and its preparation is the natural unit that is being sequenced. For example, if the library has limited complexity, then many sequences are duplicated and will result in a high duplication rate across lanes.
Sample: A single individual, such as human CEPH NA12878. Multiple libraries with different properties can be constructed from the original sample DNA source. Throughout our documentation, we treat samples as independent individuals whose genome sequence we are attempting to determine. Note that from this perspective, tumor / normal samples are different despite coming from the same individual.
Note that many GATK commands can be run at the lane level, but will give better results seeing all of the data for a single sample, or even all of the data for all samples. Unfortunately, there's a trade-off in computational cost, since running these commands across all of your data simultaneously requires much more computing power. Please see the documentation for each step to understand what is the best way to group or partition your data for that particular process.
We recommend performing variant discovery in a way that enables joint analysis of multiple samples, as laid out in our Best Practices workflow. That workflow includes a joint analysis step that empowers variant discovery by providing the ability to leverage population-wide information from a cohort of multiple sample, allowing us to detect variants with great sensitivity and genotype samples as accurately as possible. Our workflow recommendations provide a way to do this in a way that is scalable and allows incremental processing of the sequencing data.
The key point is that you don’t actually have to call variants on all your samples together to perform a joint analysis. We have developed a workflow that allows us to decouple the initial identification of potential variant sites (ie variant calling) from the genotyping step, which is the only part that really needs to be done jointly. Since GATK 3.0, you can use the HaplotypeCaller to call variants individually per-sample in
-ERC GVCF mode, followed by a joint genotyping step on all samples in the cohort, as described in this method article. This achieves what we call incremental joint discovery, providing you with all the benefits of classic joint calling (as described below) without the drawbacks.
Why "almost always"? Because some people have reported missing a small fraction of singletons (variants that are unique to individual samples) when using the new method. For most studies, this is an acceptable tradeoff (which is reduced by the availability of high quality sequencing data), but if you are very specifically looking for singletons, you may need to do some careful evaluation before committing to this method.
Until recently, three strategies were available for variant discovery in multiple samples:
- single sample calling: sample BAMs are analyzed individually, and individual call sets are combined in a downstream processing step;
- batch calling: sample BAMs are analyzed in separate batches, and batch call sets are merged in a downstream processing step;
- joint calling: variants are called simultaneously across all sample BAMs, generating a single call set for the entire cohort.
The best of these, from the point of view of variant discovery, was joint calling, because it provided the following benefits:
Batch-calling does not output a genotype call at sites where no member in the batch has evidence for a variant; it is thus impossible to distinguish such sites from locations missing data. In contrast, joint calling emits genotype calls at every site where any individual in the call set has evidence for variation.
By sharing information across all samples, joint calling makes it possible to “rescue” genotype calls at sites where a carrier has low coverage but other samples within the call set have a confident variant at that location. However this does not apply to singletons, which are unique to a single sample. To minimize the chance of missing singletons, we increase the cohort size -- so that singletons themselves have less chance of happening in the first place.
The current approaches to variant filtering (such as VQSR) use statistical models that work better with large amounts of data. Of the three calling strategies above, only joint calling provides enough data for accurate error modeling and ensures that filtering is applied uniformly across all samples.
Figure 1: Power of joint calling in finding mutations at low coverage sites. The variant allele is present in only two of the N samples, in both cases with such low coverage that the variant is not callable when processed separately. Joint calling allows evidence to be accumulated over all samples and renders the variant callable. (right) Importance of joint calling to square off the genotype matrix, using an example of two disease-relevant variants. Neither sample will have records in a variants-only output file, for different reasons: the first sample is homozygous reference while the second sample has no data. However, merging the results from single sample calling will incorrectly treat both of these samples identically as being non-informative.
There are two major problems with the joint calling strategy.
- Scaling & infrastructure
Joint calling scales very badly -- the calculations involved in variant calling (especially by methods like the HaplotypeCaller’s) become exponentially more computationally costly as you add samples to the cohort. If you don't have a lot of compute available, you run into limitations pretty quickly. Even here at Broad where we have fairly ridiculous amounts of compute available, we can't brute-force our way through the numbers for the larger cohort sizes that we're called on to handle.
- The N+1 problem
When you’re getting a large-ish number of samples sequenced (especially clinical samples), you typically get them in small batches over an extended period of time, and you analyze each batch as it comes in (whether it’s because the analysis is time-sensitive or your PI is breathing down your back). But that’s not joint calling, that’s batch calling, and it doesn’t give you the same significant gains that joint calling can give you. Unfortunately the joint calling approach doesn’t allow for incremental analysis -- every time you get even one new sample sequence, you have to re-call all samples from scratch.
The HaplotypeCaller is a more recent and sophisticated tool than the UnifiedGenotyper. Its ability to call SNPs is equivalent to that of the UnifiedGenotyper, its ability to call indels is far superior, and it is now capable of calling non-diploid samples. It also comprises several unique functionalities such as the reference confidence model (which enables efficient and incremental variant discovery on ridiculously large cohorts) and special settings for RNAseq data.
As of GATK version 3.3, we recommend using HaplotypeCaller in all cases, with no exceptions.
Caveats for older versions
If you are limited to older versions for project continuity, you may opt to use UnifiedGenotyper in the following cases:
The GATK runs natively on most if not all flavors of UNIX, which includes MacOSX, Linux and BSD. It is possible to get it running on Windows using Cygwin, but we don't provide any support nor instructions for that.
The GATK is a Java-based program, so you'll need to have Java installed on your machine. The Java version should be at 1.7 (at this time we don't officially support 1.8, and 1.6 no longer works). You can check what version you have by typing
java -version at the command line. This article has some more details about what to do if you don't have the right version. Note that at this time we only support the Sun/Oracle Java JDK; OpenJDK is not supported.
Some of the GATK tools produce plots using R, so if you want to get the plots you'll need to have R and Rscript installed, as well as several R libraries. Full details can be found in the Tutorial on installing required software.
The GATK does not have a Graphical User Interface (GUI). You don't open it by clicking on the
.jar file; you have to use the Console (or Terminal) to input commands. If this is all new to you, we recommend you first learn about that and follow some online tutorials before trying to use the GATK. It's not difficult but you'll need to learn some jargon and get used to living without a mouse. Trust us, it's a liberating experience :)
So you're going to a GATK workshop, and you've been selected to participate in a hands-on session? Fantastic! We're looking forward to walking you through some exercises that will help you master the tools. However -- in order to make the best of the time we have together, we'd like to ask you to come prepared. Specifically, if the workshop hosts are not providing machines and you have been asked to bring your own laptop, please complete the following steps:
Note that if you are a Mac user, you may need to install Apple's XCode Tools, which are free but fairly large, so plan ahead because it can take a loooong time to download them if your connection is anything less than super-fast.
This will typically be provided by email two to three weeks before the date of the workshop.
At the start of the session, we'll give you handouts with a walkthrough of the session so you can follow along and take notes (highly recommended!).
With that, you should be all set. See you soon!
VariantEval accepts two types of modules: stratification and evaluation modules.
CpG is a three-state stratification:
A CpG site is defined as a site where the reference base at a locus is a C and the adjacent reference base in the 3' direction is a G.
EvalRod is an N-state stratification, where N is the number of eval rods bound to VariantEval.
Sample is an N-state stratification, where N is the number of samples in the eval files.
Filter is a three-state stratification:
FunctionalClass is a four-state stratification:
CompRod is an N-state stratification, where N is the number of comp tracks bound to VariantEval.
Degeneracy is a six-state stratification:
See the [http://en.wikipedia.org/wiki/Genetic_code#Degeneracy Wikipedia page on degeneracy] for more information.
JexlExpression is an N-state stratification, where N is the number of JEXL expressions supplied to VariantEval. See [[Using JEXL expressions]]
Novelty is a three-state stratification:
CountVariants is an evaluation module that computes the following metrics:
|nProcessedLoci||Number of processed loci|
|nCalledLoci||Number of called loci|
|nRefLoci||Number of reference loci|
|nVariantLoci||Number of variant loci|
|variantRate||Variants per loci rate|
|variantRatePerBp||Number of variants per base|
|nSNPs||Number of snp loci|
|nInsertions||Number of insertion|
|nDeletions||Number of deletions|
|nComplex||Number of complex loci|
|nNoCalls||Number of no calls loci|
|nHets||Number of het loci|
|nHomRef||Number of hom ref loci|
|nHomVar||Number of hom var loci|
|nSingletons||Number of singletons|
|heterozygosity||heterozygosity per locus rate|
|heterozygosityPerBp||heterozygosity per base pair|
|hetHomRatio||heterozygosity to homozygosity ratio|
|indelRate||indel rate (insertion count + deletion count)|
|indelRatePerBp||indel rate per base pair|
|deletionInsertionRatio||deletion to insertion ratio|
CompOverlap is an evaluation module that computes the following metrics:
|nEvalSNPs||number of eval SNP sites|
|nCompSNPs||number of comp SNP sites|
|novelSites||number of eval sites outside of comp sites|
|nVariantsAtComp||number of eval sites at comp sites (that is, sharing the same locus as a variant in the comp track, regardless of whether the alternate allele is the same)|
|compRate||percentage of eval sites at comp sites|
|nConcordant||number of concordant sites (that is, for the sites that share the same locus as a variant in the comp track, those that have the same alternate allele)|
|concordantRate||the concordance rate|
A SNP in the detection set is said to be 'concordant' if the position exactly matches an entry in dbSNP and the allele is the same. To understand this and other output of CompOverlap, we shall examine a detailed example. First, consider a fake dbSNP file (headers are suppressed so that one can see the important things):
$ grep -v '##' dbsnp.vcf #CHROM POS ID REF ALT QUAL FILTER INFO 1 10327 rs112750067 T C . . ASP;R5;VC=SNP;VP=050000020005000000000100;WGT=1;dbSNPBuildID=132
Now, a detection set file with a single sample, where the variant allele is the same as listed in dbSNP:
$ grep -v '##' eval_correct_allele.vcf #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 001-6 1 10327 . T C 5168.52 PASS ... GT:AD:DP:GQ:PL 0/1:357,238:373:99:3959,0,4059
Finally, a detection set file with a single sample, but the alternate allele differs from that in dbSNP:
$ grep -v '##' eval_incorrect_allele.vcf #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 001-6 1 10327 . T A 5168.52 PASS ... GT:AD:DP:GQ:PL 0/1:357,238:373:99:3959,0,4059
Running VariantEval with just the CompOverlap module:
$ java -jar $STING_DIR/dist/GenomeAnalysisTK.jar -T VariantEval \ -R /seq/references/Homo_sapiens_assembly19/v1/Homo_sapiens_assembly19.fasta \ -L 1:10327 \ -B:dbsnp,VCF dbsnp.vcf \ -B:eval_correct_allele,VCF eval_correct_allele.vcf \ -B:eval_incorrect_allele,VCF eval_incorrect_allele.vcf \ -noEV \ -EV CompOverlap \ -o eval.table
We find that the eval.table file contains the following:
$ grep -v '##' eval.table | column -t CompOverlap CompRod EvalRod JexlExpression Novelty nEvalVariants nCompVariants novelSites nVariantsAtComp compRate nConcordant concordantRate CompOverlap dbsnp eval_correct_allele none all 1 1 0 1 100.00000000 1 100.00000000 CompOverlap dbsnp eval_correct_allele none known 1 1 0 1 100.00000000 1 100.00000000 CompOverlap dbsnp eval_correct_allele none novel 0 0 0 0 0.00000000 0 0.00000000 CompOverlap dbsnp eval_incorrect_allele none all 1 1 0 1 100.00000000 0 0.00000000 CompOverlap dbsnp eval_incorrect_allele none known 1 1 0 1 100.00000000 0 0.00000000 CompOverlap dbsnp eval_incorrect_allele none novel 0 0 0 0 0.00000000 0 0.00000000
As you can see, the detection set variant was listed under nVariantsAtComp (meaning the variant was seen at a position listed in dbSNP), but only the eval_correct_allele dataset is shown to be concordant at that site, because the allele listed in this dataset and dbSNP match.
TiTvVariantEvaluator is an evaluation module that computes the following metrics:
|nTi||number of transition loci|
|nTv||number of transversion loci|
|tiTvRatio||the transition to transversion ratio|
|nTiInComp||number of comp transition sites|
|nTvInComp||number of comp transversion sites|
|TiTvRatioStandard||the transition to transversion ratio for comp sites|
All analyses done with the GATK typically involve several (though not necessarily all) of the following inputs:
This article describes the corresponding file formats that are acceptable for use with the GATK.
The GATK requires the reference sequence in a single reference sequence in FASTA format, with all contigs in the same file. The GATK requires strict adherence to the FASTA standard. All the standard IUPAC bases are accepted, but keep in mind that non-standard bases (i.e. other than ACGT, such as W for example) will be ignored (i.e. those positions in the genome will be skipped).
Some users have reported having issues with reference files that have been stored or modified on Windows filesystems. The issues manifest as "10" characters (corresponding to encoded newlines) inserted in the sequence, which cause the GATK to quit with an error. If you encounter this issue, you will need to re-download a valid master copy of the reference file, or clean it up yourself.
Gzipped fasta files will not work with the GATK, so please make sure to unzip them first. Please see this article for more information on preparing FASTA reference sequences for use with the GATK.
If you are using human data, your reads must be aligned to one of the official b3x (e.g. b36, b37) or hg1x (e.g. hg18, hg19) references. The names and order of the contigs in the reference you used must exactly match that of one of the official references canonical orderings. These are defined by historical karotyping of largest to smallest chromosomes, followed by the X, Y, and MT for the b3x references; the order is thus 1, 2, 3, ..., 10, 11, 12, ... 20, 21, 22, X, Y, MT. The hg1x references differ in that the chromosome names are prefixed with "chr" and chrM appears first instead of last. The GATK will detect misordered contigs (for example, lexicographically sorted) and throw an error. This draconian approach, though unnecessary technically, ensures that all supplementary data provided with the GATK works correctly. You can use ReorderSam to fix a BAM file aligned to a missorted reference sequence.
Our Best Practice recommendation is that you use a standard GATK reference from the GATK resource bundle.
The only input format for sequence reads that the GATK itself supports is the [Sequence Alignment/Map (SAM)] format. See [SAM/BAM] for more details on the SAM/BAM format as well as Samtools and Picard, two complementary sets of utilities for working with SAM/BAM files.
If you don't find the information you need in this section, please see our FAQs on BAM files.
If you are starting out your pipeline with raw reads (typically in FASTQ format) you'll need to make sure that when you map those reads to the reference and produce a BAM file, the resulting BAM file is fully compliant with the GATK requirements. See the Best Practices documentation for detailed instructions on how to do this.
In addition to being in SAM format, we require the following additional constraints in order to use your file with the GATK:
Below is an example well-formed SAM field header and fields (with @SQ dictionary truncated to show only the first two chromosomes for brevity):
@HD VN:1.0 GO:none SO:coordinate @SQ SN:1 LN:249250621 AS:NCBI37 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:1b22b98cdeb4a9304cb5d48026a85128 @SQ SN:2 LN:243199373 AS:NCBI37 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:a0d9851da00400dec1098a9255ac712e @RG ID:ERR000162 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR000252 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR001684 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR001685 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @PG ID:GATK TableRecalibration VN:v2.2.16 CL:Covariates=[ReadGroupCovariate, QualityScoreCovariate, DinucCovariate, CycleCovariate], use_original_quals=true, defau t_read_group=DefaultReadGroup, default_platform=Illumina, force_read_group=null, force_platform=null, solid_recal_mode=SET_Q_ZERO, window_size_nqs=5, homopolymer_nback=7, except on_if_no_tile=false, pQ=5, maxQ=40, smoothing=137 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:b4eb71ee878d3706246b7c1dbef69299 @PG ID:bwa VN:0.5.5 ERR001685.4315085 16 1 9997 25 35M * 0 0 CCGATCTCCCTAACCCTAACCCTAACCCTAACCCT ?8:C7ACAABBCBAAB?CCAABBEBA@ACEBBB@? XT:A:U XN:i:4 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 RG:Z:ERR001685 NM:i:6 MD:Z:0N0N0N0N1A0A28 OQ:Z:>>:>2>>>>>>>>>>>>>>>>>>?>>>>??>???> ERR001689.1165834 117 1 9997 0 * = 9997 0 CCGATCTAGGGTTAGGGTTAGGGTTAGGGTTAGGG >7AA<@@C?@?B?B??>9?B??>A?B???BAB??@ RG:Z:ERR001689 OQ:Z:>:<<8<<<><<><><<>7<>>>?>>??>??????? ERR001689.1165834 185 1 9997 25 35M = 9997 0 CCGATCTCCCTAACCCTAACCCTAACCCTAACCCT 758A:?>>8?=@@>>?;4<>=??@@==??@?==?8 XT:A:U XN:i:4 SM:i:25 AM:i:0 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 RG:Z:ERR001689 NM:i:6 MD:Z:0N0N0N0N1A0A28 OQ:Z:;74>7><><><>>>>><:<>>>>>>>>>>>>>>>> ERR001688.2681347 117 1 9998 0 * = 9998 0 CGATCTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 5@BA@A6B???A?B??>B@B??>B@B??>BAB??? RG:Z:ERR001688 OQ:Z:=>>>><4><<?><??????????????????????
The GATK requires that the BAM file be sorted in the same order as the reference. Unfortunately, many BAM files have headers that are sorted in some other order -- lexicographical order is a common alternative. To resort the BAM file please use ReorderSam.
The GATK accept interval files for processing subsets of the genome in several different formats. Please see the FAQs on interval lists for details.
The GATK can associate arbitrary reference ordered data (ROD) files with named tracks for all tools. Some tools require specific ROD data files for processing, and developers are free to write tools that access arbitrary data sets using the ROD interface. The general ROD system has the following syntax:
name is the name in the GATK tool (like "eval" in VariantEval),
type is the type of the file, such as VCF or dbSNP, and
file is the path to the file containing the ROD data.
The GATK supports several common file formats for reading ROD data:
Note that we no longer support the PED format. See here for converting .ped files to VCF.
In GATK versions produced between September 2010 and May 2016, the GATK had a "Phone Home" usage reporting feature that sent us information about each GATK run via the Broad filesystem (within the Broad) and Amazon's S3 cloud storage service (outside the Broad). This feature was enabled by default and required a key to be disabled (for running offline or for regulatory reasons).
The Phone Home feature was removed in version 3.6. Keys are no longer necessary, so if you had one, you can stop using it. We do not expect that including Phone Home arguments in GATK command lines would cause any errors (so this should not break any scripts), but let us know if you run into any trouble.
Note that keys remain necessary for disabling Phone Home in older versions of GATK. See further below for details on how to obtain a key.
At the time, the information provided by the Phone Home feature was critical in driving improvements to the GATK:
Below are two example GATK Run Reports showing exactly what information is sent to us each time the GATK phones home.
<GATK-run-report> <id>D7D31ULwTSxlAwnEOSmW6Z4PawXwMxEz</id> <start-time>2012/03/10 20.21.19</start-time> <end-time>2012/03/10 20.21.19</end-time> <run-time>0</run-time> <walker-name>CountReads</walker-name> <svn-version>1.4-483-g63ecdb2</svn-version> <total-memory>85000192</total-memory> <max-memory>129957888</max-memory> <user-name>depristo</user-name> <host-name>10.0.1.10</host-name> <java>Apple Inc.-1.6.0_26</java> <machine>Mac OS X-x86_64</machine> <iterations>105</iterations> </GATK-run-report>
<GATK-run-report> <id>yX3AnltsqIlXH9kAQqTWHQUd8CQ5bikz</id> <exception> <message>Failed to parse Genome Location string: 20:10,000,000-10,000,001x</message> <stacktrace class="java.util.ArrayList"> <string>org.broadinstitute.sting.utils.GenomeLocParser.parseGenomeLoc(GenomeLocParser.java:377)</string> <string>org.broadinstitute.sting.utils.interval.IntervalUtils.parseIntervalArguments(IntervalUtils.java:82)</string> <string>org.broadinstitute.sting.commandline.IntervalBinding.getIntervals(IntervalBinding.java:106)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.loadIntervals(GenomeAnalysisEngine.java:618)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.initializeIntervals(GenomeAnalysisEngine.java:585)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:231)</string> <string>org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:128)</string> <string>org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:236)</string> <string>org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:146)</string> <string>org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:92)</string> </stacktrace> <cause> <message>Position: '10,000,001x' contains invalid chars.</message> <stacktrace class="java.util.ArrayList"> <string>org.broadinstitute.sting.utils.GenomeLocParser.parsePosition(GenomeLocParser.java:411)</string> <string>org.broadinstitute.sting.utils.GenomeLocParser.parseGenomeLoc(GenomeLocParser.java:374)</string> <string>org.broadinstitute.sting.utils.interval.IntervalUtils.parseIntervalArguments(IntervalUtils.java:82)</string> <string>org.broadinstitute.sting.commandline.IntervalBinding.getIntervals(IntervalBinding.java:106)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.loadIntervals(GenomeAnalysisEngine.java:618)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.initializeIntervals(GenomeAnalysisEngine.java:585)</string> <string>org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:231)</string> <string>org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:128)</string> <string>org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:236)</string> <string>org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:146)</string> <string>org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:92)</string> </stacktrace> <is-user-exception>false</is-user-exception> </cause> <is-user-exception>true</is-user-exception> </exception> <start-time>2012/03/10 20.19.52</start-time> <end-time>2012/03/10 20.19.52</end-time> <run-time>0</run-time> <walker-name>CountReads</walker-name> <svn-version>1.4-483-g63ecdb2</svn-version> <total-memory>85000192</total-memory> <max-memory>129957888</max-memory> <user-name>depristo</user-name> <host-name>10.0.1.10</host-name> <java>Apple Inc.-1.6.0_26</java> <machine>Mac OS X-x86_64</machine> <iterations>0</iterations> </GATK-run-report>
Note that as of GATK 1.5 we no longer collected information about the command-line executed, the working directory, or tmp directory.
Versions of GATK older than 3.6 attempted to "phone home" as a normal part of each run. However, we recognized that some of our users need to run the GATK with the Phone Home disabled. To enable this, we provided an option (
-et NO_ET ) in GATK 1.5 and later to disable the Phone Home feature. To use this option, you need to contact us to request a key. Instructions for doing so are below.
To obtain a GATK key, please fill out the request form.
Running the GATK with a key is simple: you just need to append a
-K your.key argument to your customary command line, where
your.key is the path to the key file you obtained from us:
java -jar dist/GenomeAnalysisTK.jar \ -T PrintReads \ -I public/testdata/exampleBAM.bam \ -R public/testdata/exampleFASTA.fasta \ -et NO_ET \ -K your.key
-K argument is only necessary when running the GATK with the
If you get an error message from the GATK saying that your key is corrupt, unreadable, or has been revoked, please apply for a new key.
If you get an error message stating that the GATK public key could not be located or read, then something is likely wrong with your build of the GATK. If you're running the binary release, try downloading it again. If you're compiling from source, try re-compiling. If all else fails, please ask for help on our community forum.
Please note that GATK-Lite was retired in February 2013 when version 2.4 was released. See the announcement here.
You probably know by now that GATK-Lite is a free-for-everyone and completely open-source version of the GATK (licensed under the original [MIT license]( http://en.wikipedia.org/wiki/MIT_License)).
But what's in the box? What can GATK-Lite do -- or rather, what can it not do that the full version (let's call it GATK-Full) can? And what does that mean exactly, in terms of functionality, reliability and power?
To really understand the differences between GATK-Lite and GATK-Full, you need some more information on how the GATK works, and how we work to develop and improve it.
As explained here, the engine handles all the common work that's related to data access, conversion and traversal, as well as high-performance computing features. The engine is supported by an infrastructure of software libraries. If the GATK was a car, that would be the engine and chassis. What we call the *tools are attached on top of that, and they provide the various analytical and processing functionalities like variant calling and base or variant recalibration. On your car, that would be headlights, airbags and so on.
We do all our development work on a single codebase. This means that everything --the engine and all tools-- is on one common workbench. There are not different versions that we work on in parallel -- that would be crazy to manage! That's why the version numbers of GATK-Lite and GATK-Full always match: if the latest GATK-Full version is numbered 2.1-13, then the latest GATK-Lite is also numbered 2.1-13.
The most important consequence of this setup is that when we make improvements to the infrastructure and engine, the same improvements will end up in GATK Lite and in GATK Full. So for the purposes of power, speed and robustness of the GATK that is determined by the engine, there is no difference between them.
For the tools, it's a little more complicated -- but not much. When we "build" the GATK binaries (the
.jar files), we put everything from the workbench into the Full build, but we only put a subset into the Lite build. Note that this Lite subset is pretty big -- it contains all the tools that were previously available in GATK 1.x versions, and always will. We also reserve the right to add previews or not-fully-featured versions of the new tools that are in Full, at our discretion, to the Lite build.
We have a new tool that performs a brand new function (which wasn't available in GATK 1.x), and we only include it in the Full build.
Reprising the car analogy, GATK-Lite and GATK-Full are like two versions of the same car -- the basic version and the fully-equipped one. They both have the exact same engine, and most of the equipment (tools) is the same -- for example, they both have the same airbag system, and they both have headlights. But there are a few important differences:
The GATK-Full car comes with a GPS (sat-nav for our UK friends), for which the Lite car has no equivalent. You could buy a portable GPS unit from a third-party store for your Lite car, but it might not be as good, and certainly not as convenient, as the Full car's built-in one.
The underlying engine is exactly the same in both GATK-Lite and GATK-Full. Most functionalities are available in both builds, performed by the same tools. Some functionalities are available in both builds, but they are performed by different tools, and the tool in the Full build is better. New, cutting-edge functionalities are only available in the Full build, and there is no equivalent in the Lite build.
We hope this clears up some of the confusion surrounding GATK-Lite. If not, please leave a comment and we'll do our best to clarify further!
One of the key challenges of working with next-gen sequence data is that input files are usually very large. We can’t just make the program open the files, load all the data into memory and perform whatever analysis is needed on all of it in one go. It’s just too much work, even for supercomputers.
Instead, we make the program cut the job into smaller tasks that the computer can easily process separately. Then we have it combine the results of each step into the final result.
Map/Reduce is the technique we use to achieve this. It consists of three steps formally called
reduce. Let’s apply it to an example case where we want to find out what is the average depth of coverage in our dataset for a certain region of the genome.
filter determines what subset of the data needs to be processed in each task. In our example, the program lists all the reference positions in our region of interest.
map applies the function, i.e. performs the analysis on each subset of data. In our example, for each position in the list, the program looks into the BAM file, pulls out the pileup of bases and outputs the depth of coverage at that position.
reducecombines the elements in the list of results output by the
mapfunction. In our example, the program takes the coverage numbers that were calculated separately for all the reference positions and calculates their average, which is the final result we want.
This may seem trivial for such a simple example, but it is a very powerful method with many advantages. Among other things, it makes it relatively easy to parallelize operations, which makes the tools run much faster on large datasets.
All the tools in the GATK are built from the ground up to take advantage of this method. That’s why we call them walkers: because they “walk” across the genome, getting things done.
Note that even though it’s not included in the Map/Reduce technique’s name, the
filter step is very important. It determines what data get presented to the tool for analysis, selecting only the appropriate data for each task and discarding anything that’s not relevant. This is a key part of the Map/Reduce technique, because that’s what makes each task “bite-sized” enough for the computer to handle easily.
Each tool has filters that are tailored specifically for the type of analysis it performs. The filters rely on traversal engines, which are little programs that are designed to “traverse” the data (i.e. walk through the data) in specific ways.
There are three major types of traversal: Locus Traversal, Read Traversal and Active Region Traversal. In our interval coverage example, the tool’s filter uses the Locus Traversal engine, which walks through the data by locus, i.e. by position along the reference genome. Because of that, the tool is classified as a Locus Walker. Similarly, the Read Traversal engine is used, you’ve guessed it, by Read Walkers.
The GATK engine comes packed with many other ways to walk through the genome and get the job done seamlessly, but those are the ones you’ll encounter most often.
GVCF stands for Genomic VCF. A GVCF is a kind of VCF, so the basic format specification is the same as for a regular VCF (see the spec documentation here), but a Genomic VCF contains extra information.
This document explains what that extra information is and how you can use it to empower your variants analyses.
What we're covering here is strictly limited to GVCFs produced by HaplotypeCaller in GATK versions 3.0 and above. The term GVCF is sometimes used simply to describe VCFs that contain a record for every position in the genome (or interval of interest) regardless of whether a variant was detected at that site or not (such as VCFs produced by UnifiedGenotyper with
--output_mode EMIT_ALL_SITES). GVCFs produced by HaplotypeCaller 3.x contain additional information that is formatted in a very specific way. Read on to find out more.
The key difference between a regular VCF and a gVCF is that the gVCF has records for all sites, whether there is a variant call there or not. The goal is to have every site represented in the file in order to do joint analysis of a cohort in subsequent steps. The records in a gVCF include an accurate estimation of how confident we are in the determination that the sites are homozygous-reference or not. This estimation is generated by the HaplotypeCaller's built-in reference model.
Note that some other tools (including the GATK's own UnifiedGenotyper) may output an all-sites VCF that looks superficially like the
BP_RESOLUTION gVCFs produced by HaplotypeCaller, but they do not provide an accurate estimate of reference confidence, and therefore cannot be used in joint genotyping analyses.
As you can see in the figure above, there are two options you can use with
BP_RESOLUTION, you get a gVCF with an individual record at every site: either a variant record, or a non-variant record. With
GVCF, you get a gVCF with individual variant records for variant sites, but the non-variant sites are grouped together into non-variant block records that represent intervals of sites for which the genotype quality (GQ) is within a certain range or band. The GQ ranges are defined in the
##GVCFBlock line of the gVCF header. The purpose of the blocks (also called banding) is to keep file size down, and there is no downside for the downstream analysis, so we do recommend using the
This is a banded gVCF produced by HaplotypeCaller with the
As you can see in the first line, the basic file format is a valid version 4.1 VCF:
##fileformat=VCFv4.1 ##ALT=<ID=NON_REF,Description="Represents any possible alternative allele at this location"> ##FILTER=<ID=LowQual,Description="Low quality"> ##FORMAT=<ID=AD,Number=.,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed"> ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)"> ##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality"> ##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype"> ##FORMAT=<ID=MIN_DP,Number=1,Type=Integer,Description="Minimum DP observed within the GVCF block"> ##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification"> ##FORMAT=<ID=SB,Number=4,Type=Integer,Description="Per-sample component statistics which comprise the Fisher's Exact Test to detect strand bias."> ##GVCFBlock=minGQ=0(inclusive),maxGQ=5(exclusive) ##GVCFBlock=minGQ=20(inclusive),maxGQ=60(exclusive) ##GVCFBlock=minGQ=5(inclusive),maxGQ=20(exclusive) ##GVCFBlock=minGQ=60(inclusive),maxGQ=2147483647(exclusive) ##INFO=<ID=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities"> ##INFO=<ID=ClippingRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref number of hard clipped bases"> ##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered"> ##INFO=<ID=DS,Number=0,Type=Flag,Description="Were any of the samples downsampled?"> ##INFO=<ID=END,Number=1,Type=Integer,Description="Stop position of the interval"> ##INFO=<ID=HaplotypeScore,Number=1,Type=Float,Description="Consistency of the site with at most two segregating haplotypes"> ##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation"> ##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as the AC), for each ALT allele, in the same order as listed"> ##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same as the AF), for each ALT allele, in the same order as listed"> ##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality"> ##INFO=<ID=MQ0,Number=1,Type=Integer,Description="Total Mapping Quality Zero Reads"> ##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities"> ##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias"> ##contig=<ID=20,length=63025520,assembly=b37> ##reference=file:///humgen/1kg/reference/human_g1k_v37.fasta
Toward the middle you see the
##GVCFBlock lines (after the
##FORMAT lines) (repeated here for clarity):
##GVCFBlock=minGQ=0(inclusive),maxGQ=5(exclusive) ##GVCFBlock=minGQ=20(inclusive),maxGQ=60(exclusive) ##GVCFBlock=minGQ=5(inclusive),maxGQ=20(exclusive)
which indicate the GQ ranges used for banding (corresponding to the boundaries
[5, 20, 60]).
You can also see the definition of the
MIN_DP annotation in the
The first thing you'll notice, hopefully, is the
<NON_REF> symbolic allele listed in every record's
ALT field. This provides us with a way to represent the possibility of having a non-reference allele at this site, and to indicate our confidence either way.
The second thing to look for is the
END tag in the
INFO field of non-variant block records. This tells you at what position the block ends. For example, the first line is a non-variant block that starts at position 20:10000000 and ends at 20:10000116.
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 20 10000000 . T <NON_REF> . . END=10000116 GT:DP:GQ:MIN_DP:PL 0/0:44:99:38:0,89,1385 20 10000117 . C T,<NON_REF> 612.77 . BaseQRankSum=0.000;ClippingRankSum=-0.411;DP=38;MLEAC=1,0;MLEAF=0.500,0.00;MQ=221.39;MQ0=0;MQRankSum=-2.172;ReadPosRankSum=-0.235 GT:AD:DP:GQ:PL:SB 0/1:17,21,0:38:99:641,0,456,691,519,1210:6,11,11,10 20 10000118 . T <NON_REF> . . END=10000210 GT:DP:GQ:MIN_DP:PL 0/0:42:99:38:0,80,1314 20 10000211 . C T,<NON_REF> 638.77 . BaseQRankSum=0.894;ClippingRankSum=-1.927;DP=42;MLEAC=1,0;MLEAF=0.500,0.00;MQ=221.89;MQ0=0;MQRankSum=-1.750;ReadPosRankSum=1.549 GT:AD:DP:GQ:PL:SB 0/1:20,22,0:42:99:667,0,566,728,632,1360:9,11,12,10 20 10000212 . A <NON_REF> . . END=10000438 GT:DP:GQ:MIN_DP:PL 0/0:52:99:42:0,99,1403 20 10000439 . T G,<NON_REF> 1737.77 . DP=57;MLEAC=2,0;MLEAF=1.00,0.00;MQ=221.41;MQ0=0 GT:AD:DP:GQ:PL:SB 1/1:0,56,0:56:99:1771,168,0,1771,168,1771:0,0,0,0 20 10000440 . T <NON_REF> . . END=10000597 GT:DP:GQ:MIN_DP:PL 0/0:56:99:49:0,120,1800 20 10000598 . T A,<NON_REF> 1754.77 . DP=54;MLEAC=2,0;MLEAF=1.00,0.00;MQ=185.55;MQ0=0 GT:AD:DP:GQ:PL:SB 1/1:0,53,0:53:99:1788,158,0,1788,158,1788:0,0,0,0 20 10000599 . T <NON_REF> . . END=10000693 GT:DP:GQ:MIN_DP:PL 0/0:51:99:47:0,120,1800 20 10000694 . G A,<NON_REF> 961.77 . BaseQRankSum=0.736;ClippingRankSum=-0.009;DP=54;MLEAC=1,0;MLEAF=0.500,0.00;MQ=106.92;MQ0=0;MQRankSum=0.482;ReadPosRankSum=1.537 GT:AD:DP:GQ:PL:SB 0/1:21,32,0:53:99:990,0,579,1053,675,1728:9,12,10,22 20 10000695 . G <NON_REF> . . END=10000757 GT:DP:GQ:MIN_DP:PL 0/0:48:99:45:0,120,1800 20 10000758 . T A,<NON_REF> 1663.77 . DP=51;MLEAC=2,0;MLEAF=1.00,0.00;MQ=59.32;MQ0=0 GT:AD:DP:GQ:PL:SB 1/1:0,50,0:50:99:1697,149,0,1697,149,1697:0,0,0,0 20 10000759 . A <NON_REF> . . END=10001018 GT:DP:GQ:MIN_DP:PL 0/0:40:99:28:0,65,1080 20 10001019 . T G,<NON_REF> 93.77 . BaseQRankSum=0.058;ClippingRankSum=-0.347;DP=26;MLEAC=1,0;MLEAF=0.500,0.00;MQ=29.65;MQ0=0;MQRankSum=-0.925;ReadPosRankSum=0.000 GT:AD:DP:GQ:PL:SB 0/1:19,7,0:26:99:122,0,494,179,515,694:12,7,4,3 20 10001020 . C <NON_REF> . . END=10001020 GT:DP:GQ:MIN_DP:PL 0/0:26:72:26:0,72,1080 20 10001021 . T <NON_REF> . . END=10001021 GT:DP:GQ:MIN_DP:PL 0/0:25:37:25:0,37,909 20 10001022 . C <NON_REF> . . END=10001297 GT:DP:GQ:MIN_DP:PL 0/0:30:87:25:0,72,831 20 10001298 . T A,<NON_REF> 1404.77 . DP=41;MLEAC=2,0;MLEAF=1.00,0.00;MQ=171.56;MQ0=0 GT:AD:DP:GQ:PL:SB 1/1:0,41,0:41:99:1438,123,0,1438,123,1438:0,0,0,0 20 10001299 . C <NON_REF> . . END=10001386 GT:DP:GQ:MIN_DP:PL 0/0:43:99:39:0,95,1226 20 10001387 . C <NON_REF> . . END=10001418 GT:DP:GQ:MIN_DP:PL 0/0:41:42:39:0,21,315 20 10001419 . T <NON_REF> . . END=10001425 GT:DP:GQ:MIN_DP:PL 0/0:45:12:42:0,9,135 20 10001426 . A <NON_REF> . . END=10001427 GT:DP:GQ:MIN_DP:PL 0/0:49:0:48:0,0,1282 20 10001428 . T <NON_REF> . . END=10001428 GT:DP:GQ:MIN_DP:PL 0/0:49:21:49:0,21,315 20 10001429 . G <NON_REF> . . END=10001429 GT:DP:GQ:MIN_DP:PL 0/0:47:18:47:0,18,270 20 10001430 . G <NON_REF> . . END=10001431 GT:DP:GQ:MIN_DP:PL 0/0:45:0:44:0,0,1121 20 10001432 . A <NON_REF> . . END=10001432 GT:DP:GQ:MIN_DP:PL 0/0:43:18:43:0,18,270 20 10001433 . T <NON_REF> . . END=10001433 GT:DP:GQ:MIN_DP:PL 0/0:44:0:44:0,0,1201 20 10001434 . G <NON_REF> . . END=10001434 GT:DP:GQ:MIN_DP:PL 0/0:44:18:44:0,18,270 20 10001435 . A <NON_REF> . . END=10001435 GT:DP:GQ:MIN_DP:PL 0/0:44:0:44:0,0,1130 20 10001436 . A AAGGCT,<NON_REF> 1845.73 . DP=43;MLEAC=2,0;MLEAF=1.00,0.00;MQ=220.07;MQ0=0 GT:AD:DP:GQ:PL:SB 1/1:0,42,0:42:99:1886,125,0,1888,126,1890:0,0,0,0 20 10001437 . A <NON_REF> . . END=10001437 GT:DP:GQ:MIN_DP:PL 0/0:44:0:44:0,0,0
Note that toward the end of this snippet, you see multiple consecutive non-variant block records. These were not merged into a single record because the sites they contain belong to different ranges of GQ (which are defined in the header).
-ERC GVCFmode, please see this companion document. For information specific to SOMATIC calls, see the MuTect documentation.
VCF stands for Variant Call Format. It is a standardized text file format for representing SNP, indel, and structural variation calls. The VCF specification used to be maintained by the 1000 Genomes Project, but its management and expansion has been taken over by the Global Alliance for Genomics and Health Data Working group file format team. The full format spec can be found in the Samtools/Hts-specs repository along with other useful specs like SAM/BAM. We highly encourage you to take a look at those documents, as they contain a lot of useful information that we don't go over in this document.
VCF is the primary (and only well-supported) format used by the GATK for variant calls. We prefer it above all others because while it can be a bit verbose, the VCF format is very explicit about the exact type and sequence of variation as well as the genotypes of multiple samples for this variation.
That being said, this highly detailed information can be challenging to understand. The information provided by the GATK tools that infer variation from high-throughput sequencing data, such as the HaplotypeCaller, is especially complex. This document describes the key features and annotations that you need to know about in order to understand VCF files output by the GATK tools.
Note that VCF files are plain text files, so you can open them for viewing or editing in any text editor, with the following caveats:
Some VCF files are very large, so your personal computer may struggle to load the whole file into memory. In such cases, you may need to use a different approach, such as using UNIX tools to access the part of the dataset that is relevant to you, or subsetting the data using tools like GATK's SelectVariants.
NEVER EDIT A VCF IN A WORD PROCESSOR SUCH AS MICROSOFT WORD BECAUSE IT WILL SCREW UP THE FORMAT! You have been warned :)
A valid VCF file is composed of two main parts: the header, and the variant call records.
The header contains information about the dataset and relevant reference sources (e.g. the organism, genome build version etc.), as well as definitions of all the annotations used to qualify and quantify the properties of the variant calls contained in the VCF file. The header of VCFs generated by GATK tools also include the command line that was used to generate them. Some other programs also record the command line in the VCF header, but not all do so as it is not required by the VCF specification. For more information about the header, see the next section.
The actual data lines will look something like this:
[HEADER LINES] #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 1 873762 . T G 5231.78 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:173,141:282:99:255,0,255 1 877664 rs3828047 A G 3931.66 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 1/1:0,105:94:99:255,255,0 1 899282 rs28548431 C T 71.77 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:1,3:4:26:103,0,26 1 974165 rs9442391 T C 29.84 LowQual [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:14,4:14:61:61,0,255
After the header lines and the field names, each line represents a single variant, with various properties of that variant represented in the columns. Note that all the lines shown in the example above describe SNPs (also called SNVs), but other variation could be described, such as indels or CNVs. See the VCF specification for details on how the various types of variations are represented. Depending on how the callset was generated, there may only be records for sites where a variant was identified, or there may also be "invariant" records, ie records for sites where no variation was identified.
You will sometimes come across VCFs that have only 8 columns, and contain no FORMAT or sample-specific information. These are called "sites-only" VCFs, and represent variation that has been observed in a population. Generally, information about the population of origin should be included in the header.
The following is a valid VCF header produced by HaplotypeCaller on an example data set (derived from our favorite test sample, NA12878). You can download similar test data from our resource bundle and try looking at it yourself!
##fileformat=VCFv4.1 ##FILTER=<ID=LowQual,Description="Low quality"> ##FORMAT=<ID=AD,Number=.,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed"> ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)"> ##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality"> ##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype"> ##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification"> ##GATKCommandLine.HaplotypeCaller=<ID=HaplotypeCaller,Version=3.4-3-gd1ac142,Date="Mon May 18 17:36:4 . . . ##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed"> ##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency, for each ALT allele, in the same order as listed"> ##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes"> ##contig=<ID=chr1,length=249250621,assembly=b37> ##reference=file:human_genome_b37.fasta
We're not showing all the lines here, but that's still a lot... so let's break it down into digestible bits. Note that the header lines are always listed in alphabetical order.
The first line:
tells you the version of the VCF specification to which the file conforms. This may seem uninteresting but it can have some important consequences for how to handle and interpret the file contents. As genomics is a fast moving field, the file formats are evolving fairly rapidly, so some of the encoding conventions change. If you run into unexpected issues while trying to parse a VCF file, be sure to check the version and the spec for any relevant format changes.
The FILTER lines tell you what filters have been applied to the data. In our test file, one filter has been applied:
Records that fail any of the filters listed here will contain the ID of the filter (here,
LowQual) in its
FILTER field (see how records are structured further below).
These lines define the annotations contained in the
INFO columns of the VCF file, which we explain further below. If you ever need to know what an annotation stands for, you can always check the VCF header for a brief explanation.
The GATKCommandLine lines contain all the parameters that went used by the tool that generated the file. Here,
GATKCommandLine.HaplotypeCaller refers to a command line invoking HaplotypeCaller. These parameters include all the arguments that the tool accepts, not just the ones specified explicitly by the user in the command line.
These contain the contig names, lengths, and which reference assembly was used with the input bam file. This can come in handy when someone gives you a callset but doesn't tell you which reference it was derived from -- remember that for most organisms, there are multiple reference assemblies, and you should always make sure to use the appropriate one!
[todo: FAQ on genome builds]
For each site record, the information is structured into columns (also called fields) as follows:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 [other samples...]
The first 8 columns of the VCF records (up to and including
INFO) represent the properties observed at the level of the variant (or invariant) site. Keep in mind that when multiple samples are represented in a VCF file, some of the site-level annotations represent a summary or average of the values obtained for that site from the different samples.
Sample-specific information such as genotype and individual sample-level annotation values are contained in the
FORMAT column (9th column) and in the sample-name columns (10th and beyond). In the example above, there is one sample called NA12878; if there were additional samples there would be additional columns to the right. Most programs order the sample columns alphabetically by sample name, but this is not always the case, so be aware that you can't depend on ordering rules for parsing VCF output!
These first 7 fields are required by the VCF format and must be present, although they can be empty (in practice, there has to be a dot, ie
. to serve as a placeholder).
CHROM and POS : The contig and genomic coordinates on which the variant occurs. Note that for deletions the position given is actually the base preceding the event.
ID: An optional identifier for the variant. Based on the contig and position of the call and whether a record exists at this site in a reference database such as dbSNP.
REF and ALT: The reference allele and alternative allele(s) observed in a sample, set of samples, or a population in general (depending how the VCF was generated). Note that REF and ALT are always given on the forward strand. For insertions, the ALT allele includes the inserted sequence as well as the base preceding the insertion so you know where the insertion is compared to the reference sequence. For deletions, the ALT allele is the base before the deletion.
QUAL: The Phred-scaled probability that a REF/ALT polymorphism exists at this site given sequencing data. Because the Phred scale is -10 * log(1-p), a value of 10 indicates a 1 in 10 chance of error, while a 100 indicates a 1 in 10^10 chance (see the FAQ article for a detailed explanation). These values can grow very large when a large amount of data is used for variant calling, so QUAL is not often a very useful property for evaluating the quality of a variant call. See our documentation on filtering variants for more information on this topic. Not to be confused with the sample-level annotation GQ; see this FAQ article for an explanation of the differences in what they mean and how they should be used.
PASSif the variant passed all filters. If the FILTER value is
., then no filtering has been applied to the records. It is extremely important to apply appropriate filters before using a variant callset in downstream analysis. See our documentation on filtering variants for more information on this topic.
This next field does not have to be present in the VCF.
=, and pairs are separated by colons, ie
;as in this example:
MQ=99.00;MQ0=0;QD=17.94. They typically summarize context information from the samples, but can also include information from other sources (e.g. population frequencies from a database resource). Some are annotated by default by the GATK tools that produce the callset, and some can be added on request. They are always defined in the VCF header, so that's an easy way to check what an annotation means if you don't recognize it. You can also find additional information on how they are calculated and how they should be interpreted in the "Annotations" section of the Tool Documentation.
At this point you've met all the fields up to INFO in this lineup:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 [other samples...]
All the rest is going to be sample-level information. Sample-level annotations are tag-value pairs, like the INFO annotations, but the formatting is a bit different. The short names of the sample-level annotations are recorded in the
FORMAT field. The annotation values are then recorded in corresponding order in each sample column (where the sample names are the
SM tags identified in the read group data). Typically, you will at minimum have information about the genotype and confidence in the genotype for the sample at each site. See the next section on genotypes for more details.
The sample-level information contained in the VCF (also called "genotype fields") may look a bit complicated at first glance, but they're actually not that hard to interpret once you understand that they're just sets of tags and values.
Let's take a look at three of the records shown earlier, simplified to just show the key genotype annotations:
1 873762 . T G [CLIPPED] GT:AD:DP:GQ:PL 0/1:173,141:282:99:255,0,255 1 877664 rs3828047 A G [CLIPPED] GT:AD:DP:GQ:PL 1/1:0,105:94:99:255,255,0 1 899282 rs28548431 C T [CLIPPED] GT:AD:DP:GQ:PL 0/1:1,3:4:26:103,0,26
Looking at that last column, here is what the tags mean:
GT : The genotype of this sample at this site. For a diploid organism, the GT field indicates the two alleles carried by the sample, encoded by a 0 for the REF allele, 1 for the first ALT allele, 2 for the second ALT allele, etc. When there's a single ALT allele (by far the more common case), GT will be either:
AD and DP : Allele depth and depth of coverage. These are complementary fields that represent two important ways of thinking about the depth of the data for this sample at this site. AD is the unfiltered allele depth, i.e. the number of reads that support each of the reported alleles. All reads at the position (including reads that did not pass the variant caller’s filters) are included in this number, except reads that were considered uninformative. Reads are considered uninformative when they do not provide enough statistical evidence to support one allele over another. DP is the filtered depth, at the sample level. This gives you the number of filtered reads that support each of the reported alleles. You can check the variant caller’s documentation to see which filters are applied by default. Only reads that passed the variant caller’s filters are included in this number. However, unlike the AD calculation, uninformative reads are included in DP. See the Tool Documentation for more details on AD (DepthPerAlleleBySample) and DP (Coverage) for more details.
PL : Normalized Phred-scaled likelihoods of the possible genotypes. For the typical case of a monomorphic site (where there is only one ALT allele) in a diploid organism, the PL field will contain three numbers, corresponding to the three possible genotypes (0/0, 0/1, and 1/1). The PL values are normalized so that the PL of the most likely genotype (assigned in the GT field) is 0 in the Phred scale (meaning its P = 1.0 in regular scale). The other values are scaled relative to this most likely genotype. Keep in mind, if you're not familiar with the statistical lingo, that when we say PL is the "likelihood of the genotype", we mean it is "the probability that the genotype is not correct". That's why the smaller the value, the better it is. [todo: PL details doc]
With that out of the way, let's interpret the genotype information for NA12878 at 1:899282.
1 899282 rs28548431 C T [CLIPPED] GT:AD:DP:GQ:PL 0/1:1,3:4:26:103,0,26
At this site, the called genotype is
GT = 0/1, which corresponds to the alleles C/T. The confidence indicated by
GQ = 26 isn't very good, largely because there were only a total of 4 reads at this site (
DP =4), 1 of which was REF (=had the reference base) and 3 of which were ALT (=had the alternate base) (indicated by
AD=1,3). The lack of certainty is evident in the PL field, where
PL(0/1) = 0 (the normalized value that corresponds to a likelihood of 1.0) as is always the case for the assigned allele, but the next PL is
PL(1/1) = 26 (which corresponds to 10^(-2.6), or 0.0025). So although we're pretty sure there's a variant at this site, there's a chance that the genotype assignment is incorrect, and that the subject may in fact not be het (heterozygous) but be may instead be hom-var (homozygous with the variant allele). But either way, it's clear that the subject is definitely not hom-ref (homozygous with the reference allele) since
PL(0/0) = 103, which corresponds to 10^(-10.3), a very small number.
No, really, don't write your own parser if you can avoid it. This is not a comment on how smart or how competent we think you are -- it's a comment on how annoyingly obtuse and convoluted the VCF format is.
Seriously. The VCF format lends itself really poorly to parsing methods like regular expressions, and we hear sob stories all the time from perfectly competent people whose home-brewed parser broke because it couldn't handle a more esoteric feature of the format. We know we broke a bunch of people's scripts when we introduced a new representation for spanning deletions in multisample callsets. OK, we ended up replacing it with a better representation a month later that was a lot less disruptive and more in line with the spirit of the specification -- but the point is, that first version was technically legal by the 4.2 spec, and that sort of thing can happen at any time. So yes, the VCF is a difficult format to work with, and one way to deal with that safely is to not home-brew parsers.
(Why are we sticking with it anyway? Because, as Winston Churchill famously put it, VCF is the worst variant call representation, except for all the others.)
A GATKReport is simply a text document that contains well-formatted, easy to read representation of some tabular data. Many GATK tools output their results as GATKReports, so it's important to understand how they are formatted and how you can use them in further analyses.
Here's a simple example:
#:GATKReport.v1.0:2 #:GATKTable:true:2:9:%.18E:%.15f:; #:GATKTable:ErrorRatePerCycle:The error rate per sequenced position in the reads cycle errorrate.61PA8.7 qualavg.61PA8.7 0 7.451835696110506E-3 25.474613284804366 1 2.362777171937477E-3 29.844949954504095 2 9.087604507451836E-4 32.875909752547310 3 5.452562704471102E-4 34.498999090081895 4 9.087604507451836E-4 35.148316651501370 5 5.452562704471102E-4 36.072234352256190 6 5.452562704471102E-4 36.121724890829700 7 5.452562704471102E-4 36.191048034934500 8 5.452562704471102E-4 36.003457059679770 #:GATKTable:false:2:3:%s:%c:; #:GATKTable:TableName:Description key column 1:1000 T 1:1001 A 1:1002 C
This report contains two individual GATK report tables. Every table begins with a header for its metadata and then a header for its name and description. The next row contains the column names followed by the data.
We provide an R library called
gsalib that allows you to load GATKReport files into R for further analysis. Here are four simple steps to getting
gsalib, installing it and loading a report.
$ R R version 2.11.0 (2010-04-22) Copyright (C) 2010 The R Foundation for Statistical Computing ISBN 3-900051-07-0 R is free software and comes with ABSOLUTELY NO WARRANTY. You are welcome to redistribute it under certain conditions. Type 'license()' or 'licence()' for distribution details. Natural language support but running in an English locale R is a collaborative project with many contributors. Type 'contributors()' for more information and 'citation()' on how to cite R or R packages in publications. Type 'demo()' for some demos, 'help()' for on-line help, or 'help.start()' for an HTML browser interface to help. Type 'q()' to quit R.
gsaliblibrary from CRAN
gsalib library is available on the Comprehensive R Archive Network, so you can just do:
From within R (we use RStudio for convenience).
In some cases you need to explicitly tell R where to find the library; you can do this as follows:
$ cat .Rprofile .libPaths("/path/to/Sting/R/")
> d = gsa.read.gatkreport("/path/to/my.gatkreport") > summary(d) Length Class Mode CountVariants 27 data.frame list CompOverlap 13 data.frame list
There has been a lot of confusion about the difference between QUAL and GQ, and we hope this FAQ will clarify the difference.
The basic difference is that QUAL refers to the variant site whereas GQ refers to a specific sample's GT.
QUAL tells you how confident we are that there is some kind of variation at a given site. The variation may be present in one or more samples.
QUAL (or more importantly, its normalized form, QD) is mostly useful in multisample context. When you are recalibrating a cohort callset, you're going to be looking exclusively at site-level annotations like QD, because at that point what you're looking for is evidence of variation overall. That way you don't rely too much on individual sample calls, which are less robust.
In fact, many cohort studies don't even really care about individual genotype assignments, so they only use site annotations for their entire analysis.
Conversely, QUAL may seem redundant if you have only one sample. Especially if it has a good GQ (and more importantly, well separated PLs) then admittedly you don't really need to look at the QUAL -- you know what you have. If the GQ is not good, you can typically rely on the PLs to tell you whether you do probably have a variant, but we're just not sure if it's het or hom-var. If hom-ref is also a possibility, the call may be a potential false positive.
That said, it is more effective to filter on site-level annotations first, then refine and filter genotypes as appropriate. That's the workflow we recommend, based on years of experience doing this at fairly large scales...
This document describes how GATK commands are structured and how to add arguments to basic command examples.
Commands for GATK always follow the same basic syntax:
java [Java arguments] -jar GenomeAnalysisTK.jar [GATK arguments]
The core of the command is
java -jar GenomeAnalysisTK.jar, which starts up the GATK program in a Java Virtual Machine (JVM). Any additional java-specific arguments (such as -Xmx to increase memory allocation) should be inserted between
-jar, like this:
java -Xmx4G -jar GenomeAnalysisTK.jar [GATK arguments]
The order of arguments between
-jar is not important.
There are two universal arguments that are required for every GATK command (with very few exceptions, the
-R for Reference (e.g.
-R human_b37.fasta) and
-T for Tool name (e.g.
Additional arguments fall in two categories:
Engine arguments like
-L (for specifying a list of intervals) which can be given to all tools and are technically optional but may be effectively required at certain steps for specific analytical designs (e.g. the
-L argument for calling variants on exomes);
-I(to provide an input file containing sequence reads to tools that process BAM files) or optional, like
-alleles(to provide a list of known alleles for genotyping).
The ordering of GATK arguments is not important, but we recommend always passing the tool name (
-T) and reference (
-R) first for consistency. It is also a good idea to consistently order arguments by some kind of logic in order to make it easy to compare different commands over the course of a project. It’s up to you to choose what that logic should be.
All available engine and tool-specific arguments are listed in the tool documentation section. Arguments typically have both a long name (prefixed by
--) and a short name (prefixed by
-). The GATK command line parser recognizes both equally, so you can use whichever you prefer, depending on whether you prefer commands to be more verbose or more succinct.
Finally, a note about flags. Flags are arguments that have boolean values, i.e. TRUE or FALSE. They are typically used to enable or disable specific features; for example,
--keep_program_records will make certain GATK tools output additional information in the BAM header that would be omitted otherwise. In GATK, all flags are set to FALSE by default, so if you want to set one to TRUE, all you need to do is add the flag name to the command. You don't need to specify an actual value.
This is a very simple command that runs HaplotypeCaller in default mode on a single input BAM file containing sequence data and outputs a VCF file containing raw variants.
java -Xmx4G -jar GenomeAnalysisTK.jar -R human_b37.fasta -T HaplotypeCaller -I sample1.bam -o raw_variants.vcf
If the data is from exome sequencing, we should additionally provide the exome targets using the
java -Xmx4G -jar GenomeAnalysisTK.jar -R human_b37.fasta -T HaplotypeCaller -I sample1.bam -o raw_variants.vcf -L exome_intervals.list
If we just want to genotype specific sites of interest using known alleles based on results from a previous study, we can change the HaplotypeCaller’s genotyping mode using
-gt_mode, provide those alleles using
-alleles, and restrict the analysis to just those sites using
java -Xmx4G -jar GenomeAnalysisTK.jar -R human_b37.fasta -T HaplotypeCaller -I sample1.bam -o raw_variants.vcf -L known_alleles.vcf -alleles known_alleles.vcf -gt_mode GENOTYPE_GIVEN_ALLELES
For more examples of commands and for specific tool commands, see the tool documentation section.
Most sequencing providers generate FASTQ files with the raw unmapped read sequences, so that is the most common form in which the data is input into the mapping step of the pre-processing pipeline. This is not ideal because among other flaws, much of the metadata associated with sequencing runs cannot be stored in FASTQ files, unlike BAM files which can store more information. See this blog post for an overview of the many problems associated with the FASTQ format.
At the Broad Institute, we generate unmapped BAM (uBAM) files directly from the Illumina basecalls in order to keep all metadata in once place, and we do not write the data to FASTQ files at any point. This involves a slightly more complex workflow than is shown in the general Best Practices diagram. See this presentation for more details of how this works.
In case you're wondering, we still show the FASTQ-based workflow as the default in most of our documentation because it is by far the most commonly-used workflow, and we want to keep the documentation accessible for our more novice users.
Each tool uses known sites differently, but what is common to all is that they use them to help distinguish true variants from false positives, which is very important to how these tools work. If you don't provide known sites, the statistical analysis of the data will be skewed, which can dramatically affect the sensitivity and reliability of the results.
In the variant calling pipeline, the only tools that do not strictly require known sites are UnifiedGenotyper and HaplotypeCaller.
If you're working on human genomes, you're in luck. We provide sets of known sites in the human genome as part of our resource bundle, and we can give you specific Best Practices recommendations on which sets to use for each tool in the variant calling pipeline. See the next section for details.
If you're working on genomes of other organisms, things may be a little harder -- but don't panic, we'll try to help as much as we can. We've started a community discussion in the forum on What are the standard resources for non-human genomes? in which we hope people with non-human genomics experience will share their knowledge.
And if it turns out that there is as yet no suitable set of known sites for your organisms, here's how to make your own for the purposes of BaseRecalibration: First, do an initial round of SNP calling on your original, unrecalibrated data. Then take the SNPs that you have the highest confidence in and use that set as the database of known SNPs by feeding it as a VCF file to the base quality score recalibrator. Finally, do a real round of SNP calling with the recalibrated data. These steps could be repeated several times until convergence. Good luck!
Some experimentation will be required to figure out the best way to find the highest confidence SNPs for use here. Perhaps one could call variants with several different calling algorithms and take the set intersection. Or perhaps one could do a very strict round of filtering and take only those variants which pass the test.
|Tool||dbSNP 129||dbSNP >132||Mills indels||1KG indels||HapMap||Omni|
These tools require known indels passed with the
-known argument to function properly. We use both the following files:
This tool requires known SNPs and indels passed with the
-knownSites argument to function properly. We use all the following files:
These tools do NOT require known sites, but if SNPs are provided with the
-dbsnp argument they will use them for variant annotation. We use this file:
For VariantRecalibrator, please see the FAQ article on VQSR training sets and arguments.
This tool requires known SNPs passed with the
-dbsnp argument to function properly. We use the following file:
The answer depends on what tool we're talking about, and whether we're considering variant discovery or variant manipulation.
GATK variant manipulation tools are able to recognize the following types of alleles:
<NON-REF>allele used in GVCFs produced by HaplotypeCaller, the
*allele used to signify the presence of a spanning deletion, or undefined events like a very large allele or one that's fuzzy and not fully modeled; i.e. there's some event going on here but we don't know what exactly)
Note that SelectVariants, the GATK tool most used for VCF subsetting operations, discriminates strictly between these categories. This means that if you use for example
INDEL to pull out indels, it will only select pure INDEL records, excluding any MIXED records that might include a SNP allele in addition to the insertion or deletion alleles of interest. To include those you would have to also specify
selectType MIXED in the same command.
The HaplotypeCaller is a sophisticated variant caller that can call different types of variants at the same time. So in addition to SNPs and indels, it is capable of emitting mixed records by default, as well as symbolic representations for e.g. spanning deletions. It does emit physical phasing information, but in its current version, HC is not able to emit MNPs. If you would like to combine contiguous SNPs into MNPs, you will need to use the ReadBackedPhasing tool with the MNP merging function activated. See the tool documentation for details. Our older (and now deprecated) variant caller, UnifiedGenotyper, was even more limited. It only called SNPs and indels, and did so separately (even if you ran in calling mode BOTH, the program performed separate calling operations internally) so it was not able to recognize that SNPs and Indels should be emitted together as a joint record when they occur at the same site.
The general release version of GATK is currently not able to detect SVs (structural variations) or CNVs (copy number variations). However, the alpha version of GATK 4 (the next generation of GATK tools) includes tools for performing CNV (copy number variation) analysis in exome data. Let us know if you're interested in trying them out by commenting on this article in the forum.
There is also a third-party software package called GenomeSTRiP built on top of GATK that provides SV (structural variation) analysis capabilities.
NOTE: we are currently working on overhauling the bundle to 1) add support for Hg38 and 2) document the provenance of the resource files more fully.
See the Resource Bundle page. In a nutshell, there's a Google Cloud bucket and an FTP server. We only provide Hg38 resources in the cloud bucket; the rest is only available through the FTP server.
In the cloud All the resource files needed for Best Practices short variant discovery in whole-genome sequencing data (WGS). Exome files coming soon. Detailed documentation in progress (ToW Aug 2016), stay tuned for announcements.
Additionally, these files all have supplementary indices, statistics, and other QC data available.
Includes the UCSC-style hg19 reference along with all lifted over VCF files.
Includes the UCSC-style hg18 reference along with all lifted over VCF files. The refGene track and BAM files are not available. We only provide data files for this genome-build that can be lifted over "easily" from our master b37 repository. Sorry for whatever inconvenience that this might cause.
Also includes a chain file to lift over to b37.
Includes the 1000 Genomes pilot b36 formatted reference sequence (human_b36_both.fasta) along with all lifted over VCF files. The refGene track and BAM files are not available. We only provide data files for this genome-build that can be lifted over "easily" from our master b37 repository. Sorry for whatever inconvenience that this might cause.
Also includes a chain file to lift over to b37.
The -L argument (short for --intervals) enables you to restrict your analysis to specific intervals instead of running over the whole genome. Using this argument can have important consequences for performance and/or results. Here, we present some guidelines for using it appropriately depending on your experimental design.
- Whole genome analysis: no need to include intervals
- Whole exome analysis: you need to provide the list of capture targets (typically genes/exons)
- Small targeted experiment: you need to provide the targeted interval(s)
- Troubleshooting: you can run on a specific interval to test parameters or create a data snippet
Whatever you end up using -L for, keep this in mind: for tools that output a bam or VCF file, the output file will only contain data from the intervals specified by the -L argument. To be clear, we do not recommend using -L with tools that output a bam file since doing so will omit some data from the output.
-L 20 (for chromosome 20 in b37/b39 build)
-L chr20:1-100 (for chromosome 20 positions 1-100 in hg18/hg19 build)
- For example,
HLA-A*01:01:01:01 is a new contig in GRCh38. The colons are a new feature of contig naming for GRCh38 from prior assemblies. This has implications for using the
-L option of GATK as the option also uses the colon as a delimiter to distinguish between contig and genomic coordinates.
- When defining coordinates of interest for a contig, e.g. positions 1-100 for chr1, we would use
-L chr1:1-100. This also works for our HLA contig, e.g.
- However, when passing in an entire contig, for contigs with colons in the name, you must add
:1+ to the end of the chromosome name as shown below. This ensures that portions of the contig name are appropriately identified as part of the contig name and not genomic coordinates.
It is not necessary to use -L in whole genome analysis. You should be interested in the whole genome!
Nevertheless, in some cases, you may want to mask out certain contigs (e.g. chrY or non-chromosome contigs) or regions (e.g. centromere). You can do this with -XL, which does the exact opposite of -L; it excludes the provided intervals.
By definition, exome sequencing data doesn’t cover the entire genome, so many analyses can be restricted to just the capture targets (genes or exons) to save processing time. There are even some analyses which should be restricted to the capture targets because failing to do so can lead to suboptimal results.
Note that we recommend adding some “padding” to the intervals in order to include the flanking regions (typically ~100 bp). No need to modify your target list; you can have the GATK engine do it for you automatically using the interval padding argument. This is not required, but if you do use it, you should do it consistently at all steps where you use -L.
Below is a step-by-step breakdown of the Best Practices workflow, with a detailed explanation of why -L should or shouldn’t be used with each tool.
|Tool||-L?||Why / why not|
|RealignerTargetCreator||YES||Faster since RTC will only look for regions that need to be realigned within the input interval; no time wasted on the rest.|
|IndelRealigner||NO||IR will only try to realign the regions output from RealignerTargetCreator, so there is nothing to be gained by providing the capture targets.|
|BaseRecalibrator||YES||This excludes off-target sequences and sequences that may be poorly mapped, which have a higher error rate. Including them could lead to a skewed model and bad recalibration.|
|PrintReads||NO||Output is a bam file; using -L would lead to lost data.|
|UnifiedGenotyper/Haplotype Caller||YES||We’re only interested in making calls in exome regions; the rest is a waste of time & includes lots of false positives.|
|Next steps||NO||No need since subsequent steps operate on the callset, which was restricted to the exome at the calling step.|
The same guidelines as for whole exome analysis apply except you do not run BQSR on small datasets.
You can go crazy with -L while troubleshooting! For example, you can just provide an interval at the command line, and the output file will contain the data from that interval.This is really useful when you’re trying to figure out what’s going on in a specific interval (e.g. why HaplotypeCaller is not calling your favorite indel) or what would be the effect of changing a parameter (e.g. what happens to your indel call if you increase the value of -minPruning). This is also what you’d use to generate a file snippet to send us as part of a bug report (except that never happens because GATK has no bugs, ever).
From the NCBI RefSeq website
The Reference Sequence (RefSeq) collection aims to provide a comprehensive, integrated, non-redundant, well-annotated set of sequences, including genomic DNA, transcripts, and proteins. RefSeq is a foundation for medical, functional, and diversity studies; they provide a stable reference for genome annotation, gene identification and characterization, mutation and polymorphism analysis (especially RefSeqGene records), expression studies, and comparative analyses.
The GATK uses RefSeq in a variety of walkers, from indel calling to variant annotations. There are many file format flavors of ReqSeq; we've chosen to use the table dump available from the UCSC genome table browser.
Go to the UCSC genome table browser. There are many output options, here are the changes that you'll need to make:
clade: Mammal genome: Human assembly: ''choose the appropriate assembly for the reference you're using'' group: Genes abd Gene Prediction Tracks track: RefSeq Genes table: refGene region: ''choose the genome option''
Choose a good output filename, something like
geneTrack.refSeq, and click the
get output button. You now have your initial RefSeq file, which will not be sorted, and will contain non-standard contigs. To run with the GATK, contigs other than the standard 1-22,X,Y,MT must be removed, and the file sorted in karyotypic order.
You can provide your RefSeq file to the GATK like you would for any other ROD command line argument. The line would look like the following:
Using the filename from above.
The GATK automatically adjusts the start and stop position of the records from zero-based half-open intervals (UCSC standard) to one-based closed intervals.
The first 19 bases in Chromosome one: Chr1:0-19 (UCSC system) Chr1:1-19 (GATK)
All of the GATK output is also in this format, so if you're using other tools or scripts to process RefSeq or GATK output files, you should be aware of this difference.
We distinguish "Classic GATK" (major versions 1 through 3) and GATK 4, the next generation of GATK tools.
This repository contains the code corresponding to the core GATK development framework, including the GATK engine and many utilities, which third-party developers can use to develop their own GATK-based analysis tools. Be advised however that support for development using this framework is being discontinued.
This repository contains the code corresponding to the
GenomeAnalysisTK.jar file that we distribute to our users, containing the GATK engine and all analysis tools.
This includes the code in broadgsa/gatk under the MIT license, plus tools and utilities that are under a more restrictive license that prohibits commercial/for-profit use. Anyone interested in accessing the protected code for commercial/for-profit purposes should contact our licensing department (email@example.com) to inquire about licensing terms.
The code for GATK 4+, currently available as an alpha preview, is accessible through two publicly accessible Github repositories: broadinstitute/gatk and broadinstitute/gatk-protected. The division is also based on having two different licenses, like Classic GATK, but in this case the repositories are complementary; there is no code shared between them.
This repository contains the code corresponding to the core GATK 4+ development framework, including the new GATK engine and many utilities, which third-party developers can use to develop their own GATK-based analysis tools. We encourage developers to use this new framework for development and we welcome feedback regarding features and development support.
This repository contains the code for key analysis tools that are covered under a more restrictive license that prohibits commercial/for-profit use. Anyone interested in accessing the protected code for commercial/for-profit purposes should contact our licensing department (firstname.lastname@example.org) to inquire about licensing terms.
We have sequenced at the Broad Institute and released to the 1000 Genomes Project the following datasets for the three members of the CEU trio (NA12878, NA12891 and NA12892):
This is better data to work with than the original DePristo et al. BAMs files, so we recommend you download and analyze these files if you are looking for complete, large-scale data sets to evaluate the GATK or other tools.
Here's the rough library properties of the BAMs:
These data files can be downloaded from the 1000 Genomes DCC
Here are the datasets we used in the GATK paper cited below.
DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D and Daly, M (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics. 43:491-498.
Some of the BAM and VCF files are currently hosted by the NCBI: ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/technical/working/20101201_cg_NA12878/
-- targetsused in the analysis of the exome capture data
Please note that we have not collected the indel calls for the paper, as these are only used for filtering SNPs near indels. If you want to call accurate indels, please use the new GATK indel caller in the Unified Genotyper.
Both the GATK and the sequencing technologies have improved significantly since the analyses performed in this paper.
If you are conducting a review today, we would recommend that the newest version of the GATK, which performs much better than the version described in the paper. Moreover, we would also recommend one use the newest version of Crossbow as well, in case they have improved things. The GATK calls for NA12878 from the paper (above) will give one a good idea what a good call set looks like whole-genome or whole-exome.
Obviously, this was an annoyance for us as well, as it would have been nice to use a state-of-the-art data set for the WEx. But we decided to freeze the data used for analysis to actually finish this paper.
If you want the raw, machine output for the data analyzed in the GATK framework paper, obtain the raw BAM files above and convert them from SAM to FASTQ using the Picard tool SamToFastq.
There are two types of GATK tools that are able to use pedigree (family structure) information:
The two variant callers (HaplotypeCaller and the deprecated UnifiedGenotyper) as well as VariantAnnotator and GenotypeGVCFs are all able to use pedigree information if you request an annotation that involves population structure (e.g. Inbreeding Coefficient). To be clear though, the pedigree information is not used during the variant calling process; it is only used during the annotation step at the end.
If you already have VCF files that were called without pedigree information, and you want to add pedigree-related annotations (e.g to use Variant Quality Score Recalibration (VQSR) with the InbreedingCoefficient as a feature annotation), don't panic. Just run the latest version of the VariantAnnotator to re-annotate your variants, requesting any missing annotations, and make sure you pass your PED file to the VariantAnnotator as well. If you forget to provide the pedigree file, the tool will run successfully but pedigree-related annotations may not be generated (this behavior is different in some older versions).
The PED files used as input for these tools are based on PLINK pedigree files. The general description can be found here.
For these tools, the PED files must contain only the first 6 columns from the PLINK format PED file, and no alleles, like a FAM file in PLINK.
This document describes the resource datasets and arguments that we recommend for use in the two steps of VQSR (i.e. the successive application of VariantRecalibrator and ApplyRecalibration), based on our work with human genomes, to comply with the GATK Best Practices. The recommendations detailed in this document take precedence over any others you may see elsewhere in our documentation (e.g. in Tutorial articles, which are only meant to illustrate usage, or in past presentations, which may be out of date).
The document covers:
These recommendations are valid for use with calls generated by both the UnifiedGenotyper and HaplotypeCaller. In the past we made a distinction in how we processed the calls from these two callers, but now we treat them the same way. These recommendations will probably not work properly on calls generated by other (non-GATK) callers.
Note that VQSR must be run twice in succession in order to build a separate error model for SNPs and INDELs (see the VQSR documentation for more details).
The human genome training, truth and known resource datasets mentioned in this document are all available from our resource bundle.
If you are working with non-human genomes, you will need to find or generate at least truth and training resource datasets with properties corresponding to those described below. To generate your own resource set, one idea is to first do an initial round of SNP calling and only use those SNPs which have the highest quality scores. These sites which have the most confidence are probably real and could be used as truth data to help disambiguate the rest of the variants in the call set. Another idea is to try using several SNP callers in addition to the UnifiedGenotyper or HaplotypeCaller, and use those sites which are concordant between the different methods as truth data. In either case, you'll need to assign your set a prior likelihood that reflects your confidence in how reliable it is as a truth set. We recommend Q10 as a starting value, which you can then experiment with to find the most appropriate value empirically. There are many possible avenues of research here. Hopefully the model reporting plots that are generated by the recalibration tools will help facilitate this experimentation.
True sites training resource: HapMap
This resource is a SNP call set that has been validated to a very high degree of confidence. The program will consider that the variants in this resource are representative of true sites (truth=true), and will use them to train the recalibration model (training=true). We will also use these sites later on to choose a threshold for filtering variants based on sensitivity to truth sites. The prior likelihood we assign to these variants is Q15 (96.84%).
True sites training resource: Omni
This resource is a set of polymorphic SNP sites produced by the Omni geno- typing array. The program will consider that the variants in this resource are representative of true sites (truth=true), and will use them to train the recalibration model (training=true). The prior likelihood we assign to these variants is Q12 (93.69%).
Non-true sites training resource: 1000G
This resource is a set of high-confidence SNP sites produced by the 1000 Genomes Project. The program will consider that the variants in this re- source may contain true variants as well as false positives (truth=false), and will use them to train the recalibration model (training=true). The prior likelihood we assign to these variants is Q10 (90%).
Known and true sites training resource: Mills
This resource is an Indel call set that has been validated to a high degree of confidence. The program will consider that the variants in this resource are representative of true sites (truth=true), and will use them to train the recalibration model (training=true). The prior likelihood we assign to these variants is Q12 (93.69%).
Some of the annotations included in the recommendations given below might not be the best for your particular dataset. In particular, the following caveats apply:
Depth of coverage (the DP annotation invoked by Coverage) should not be used when working with exome datasets since there is extreme variation in the depth to which targets are captured! In whole genome experiments this variation is indicative of error but that is not the case in capture experiments.
You may have seen HaplotypeScore mentioned in older documents. That is a statistic produced by UnifiedGenotyper that should only be used if you called your variants with UG. This statistic isn't produced by the HaplotypeCaller because that mathematics is already built into the likelihood function itself when calling full haplotypes with HC.
In our testing we've found that in order to achieve the best exome results one needs to use an exome SNP and/or indel callset with at least 30 samples. For users with experiments containing fewer exome samples there are several options to explore:
Add additional samples for variant calling, either by sequencing additional samples or using publicly available exome bams from the 1000 Genomes Project (this option is used by the Broad exome production pipeline). Be aware that you cannot simply add VCFs from the 1000 Genomes Project. You must either call variants from the original BAMs jointly with your own samples, or (better) use the reference model workflow to generate GVCFs from the original BAMs, and perform joint genotyping on those GVCFs along with your own samples' GVCFs with GenotypeGVCFs.
--maxGaussians 4to your command line, for example). You should only do this if you are working with a non-model organism for which there are no available genomes or exomes that you can use to supplement your own cohort.
The variant quality score recalibrator builds an adaptive error model using known variant sites and then applies this model to estimate the probability that each variant is a true genetic variant or a machine artifact. One major improvement from previous recommended protocols is that hand filters do not need to be applied at any point in the process now. All filtering criteria are learned from the data itself.
This is the first part of the VariantRecalibrator command line, to which you need to add either the SNP-specific recommendations or the indel-specific recommendations given further below.
java -Xmx4g -jar GenomeAnalysisTK.jar \ -T VariantRecalibrator \ -R path/to/reference/human_g1k_v37.fasta \ -input raw.input.vcf \ -recalFile path/to/output.recal \ -tranchesFile path/to/output.tranches \ -nt 4 \ [SPECIFY TRUTH AND TRAINING SETS] \ [SPECIFY WHICH ANNOTATIONS TO USE IN MODELING] \ [SPECIFY WHICH CLASS OF VARIATION TO MODEL] \
For SNPs we use both HapMap v3.3 and the Omni chip array from the 1000 Genomes Project as training data. In addition we take the highest confidence SNPs from the project's callset. These datasets are available in the GATK resource bundle.
-resource:hapmap,known=false,training=true,truth=true,prior=15.0 hapmap_3.3.b37.sites.vcf \ -resource:omni,known=false,training=true,truth=true,prior=12.0 1000G_omni2.5.b37.sites.vcf \ -resource:1000G,known=false,training=true,truth=false,prior=10.0 1000G_phase1.snps.high_confidence.vcf \ -resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.b37.vcf \ -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR -an DP -an InbreedingCoeff \ -mode SNP \
Please note that these recommendations are formulated for whole-genome datasets. For exomes, we do not recommend using DP for variant recalibration (see below for details of why).
Note also that, for the above to work, the input vcf needs to be annotated with the corresponding values (QD, FS, DP, etc.). If any of these values are somehow missing, then VariantAnnotator needs to be run first so that VariantRecalibration can run properly.
Also, using the provided sites-only truth data files is important here as parsing the genotypes for VCF files with many samples increases the runtime of the tool significantly.
You may notice that these recommendations no longer include the
--numBadVariants argument. That is because we have removed this argument from the tool, as the VariantRecalibrator now determines the number of variants to use for modeling "bad" variants internally based on the data.
When modeling indels with the VQSR we use a training dataset that was created at the Broad by strictly curating the (Mills, Devine, Genome Research, 2011) dataset as as well as adding in very high confidence indels from the 1000 Genomes Project. This dataset is available in the GATK resource bundle.
--maxGaussians 4 \ -resource:mills,known=false,training=true,truth=true,prior=12.0 Mills_and_1000G_gold_standard.indels.b37.sites.vcf \ -resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.b37.vcf \ -an QD -an DP -an FS -an SOR -an ReadPosRankSum -an MQRankSum -an InbreedingCoeff \ -mode INDEL \
Note that indels use a different set of annotations than SNPs. Most annotations related to mapping quality have been removed since there is a conflation with the length of an indel in a read and the degradation in mapping quality that is assigned to the read by the aligner. This covariation is not necessarily indicative of being an error in the same way that it is for SNPs.
You may notice that these recommendations no longer include the
--numBadVariants argument. That is because we have removed this argument from the tool, as the VariantRecalibrator now determines the number of variants to use for modeling "bad" variants internally based on the data.
The power of the VQSR is that it assigns a calibrated probability to every putative mutation in the callset. The user is then able to decide at what point on the theoretical ROC curve their project wants to live. Some projects, for example, are interested in finding every possible mutation and can tolerate a higher false positive rate. On the other hand, some projects want to generate a ranked list of mutations that they are very certain are real and well supported by the underlying data. The VQSR provides the necessary statistical machinery to effectively apply this sensitivity/specificity tradeoff.
This is the first part of the ApplyRecalibration command line, to which you need to add either the SNP-specific recommendations or the indel-specific recommendations given further below.
java -Xmx3g -jar GenomeAnalysisTK.jar \ -T ApplyRecalibration \ -R reference/human_g1k_v37.fasta \ -input raw.input.vcf \ -tranchesFile path/to/input.tranches \ -recalFile path/to/input.recal \ -o path/to/output.recalibrated.filtered.vcf \ [SPECIFY THE DESIRED LEVEL OF SENSITIVITY TO TRUTH SITES] \ [SPECIFY WHICH CLASS OF VARIATION WAS MODELED] \
For SNPs we used HapMap 3.3 and the Omni 2.5M chip as our truth set. We typically seek to achieve 99.5% sensitivity to the accessible truth sites, but this is by no means universally applicable: you will need to experiment to find out what tranche cutoff is right for your data. Generally speaking, projects involving a higher degree of diversity in terms of world populations can expect to achieve a higher truth sensitivity than projects with a smaller scope.
--ts_filter_level 99.5 \ -mode SNP \
For indels we use the Mills / 1000 Genomes indel truth set described above. We typically seek to achieve 99.0% sensitivity to the accessible truth sites, but this is by no means universally applicable: you will need to experiment to find out what tranche cutoff is right for your data. Generally speaking, projects involving a higher degree of diversity in terms of world populations can expect to achieve a higher truth sensitivity than projects with a smaller scope.
--ts_filter_level 99.0 \ -mode INDEL \
As featured in this forum question.
Two main things account for these kinds of differences, both linked to default behaviors of the tools:
The tools downsample to different depths of coverage
In both cases, you can end up looking at different sets or numbers of reads, which causes some of the annotation values to be different. It's usually not a cause for alarm. Remember that many of these annotations should be interpreted relatively, not absolutely.