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**BETA** Funcotator

Functional Annotator

Category Variant Evaluation and Refinement


Overview

Funcotator (FUNCtional annOTATOR) analyzes given variants for their function (as retrieved from a set of data sources) and produces the analysis in a specified output file.

This tool is a functional annotation tool that allows a user to add annotations to called variants based on a set of data sources, each with its own matching criteria. Data sources are expected to be in folders that are specified as input arguments. While multiple data source folders can be specified, no two data sources can have the same name.

Data Source Folders

In each main data source folder, there should be sub-directories for each individual data source, with further sub-directories for a specific reference (i.e. hg19 or hg38). In the reference-specific data source directory, there is a configuration file detailing information about the data source and how to match it to a variant. This configuration file is required.

An example of a data source directory is the following:

         dataSourcesFolder/
              Data_Source_1/
                  hg19
                      data_source_1.config
                      data_source_1.data.file.one
                      data_source_1.data.file.two
                      data_source_1.data.file.three
                      ...
                   hg38
                      data_source_1.config
                      data_source_1.data.file.one
                      data_source_1.data.file.two
                      data_source_1.data.file.three
                      ...
              Data_Source_2/
                  hg19
                      data_source_2.config
                      data_source_2.data.file.one
                      data_source_2.data.file.two
                      data_source_2.data.file.three
                      ...
                   hg38
                      data_source_2.config
                      data_source_2.data.file.one
                      data_source_2.data.file.two
                      data_source_2.data.file.three
                      ...
               ...
     
A gzip of data source files is provided here: ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/funcotator/.

User-Defined Data Sources

Users can define their own data sources by creating a new correctly-formatted data source sub-directory in the main data sources folder. In this sub-directory, the user must create an additional folder for the reference for which the data source is valid. If the data source is valid for multiple references, then multiple reference folders should be created. Inside each reference folder, the user should place the file(s) containing the data for the data source. Additionally the user must create a configuration file containing metadata about the data source.

There are several formats allowed for data sources, however the two most useful are arbitrarily separated value (XSV) files, such as comma-separated value (CSV), tab-separated value (TSV). These files contain a table of data that can be matched to a variant by gene name, transcript ID, or genome position. In the case of gene name and transcript ID, one column must contain the gene name or transcript ID for each row's data.

  • For gene name, when a variant is annotated with a gene name that exactly matches an entry in the gene name column for a row, that row's other fields will be added as annotations to the variant.
  • For transcript ID, when a variant is annotated with a transcript ID that exactly matches an entry in the transcript ID column for a row, that row's other fields will be added as annotations to the variant.
  • For genome position, one column must contain the contig ID, another column must contain the start position (1-based, inclusive), and a column must contain the stop position (1-based, inclusive). The start and stop columns may be the same column. When a variant is annotated with a genome position that overlaps an entry in the three genome position columns for a row, that row's other fields will be added as annotations to the variant.

Configuration File Format

The configuration file is a standard Java properties-style configuration file with key-value pairs. This file name must end in .config.

The following is an example of a genome position XSV configuration file (for the ORegAnno data source):

         name = Oreganno
         version = 20160119
         src_file = oreganno.tsv
         origin_location = http://www.oreganno.org/dump/ORegAnno_Combined_2016.01.19.tsv
         preprocessing_script = getOreganno.py

         # Supported types:
         # simpleXSV    -- Arbitrary separated value table (e.g. CSV), keyed off Gene Name OR Transcript ID
         # locatableXSV -- Arbitrary separated value table (e.g. CSV), keyed off a genome location
         # gencode      -- Custom datasource class for GENCODE
         # cosmic       -- Custom datasource class for COSMIC
         type = locatableXSV

         # Required field for GENCODE files.
         # Path to the FASTA file from which to load the sequences for GENCODE transcripts:
         gencode_fasta_path =

         # Required field for simpleXSV files.
         # Valid values:
         #     GENE_NAME
         #     TRANSCRIPT_ID
         xsv_key =

         # Required field for simpleXSV files.
         # The 0-based index of the column containing the key on which to match
         xsv_key_column =

         # Required field for simpleXSV AND locatableXSV files.
         # The delimiter by which to split the XSV file into columns.
         xsv_delimiter = \t

         # Required field for simpleXSV files.
         # Whether to permissively match the number of columns in the header and data rows
         # Valid values:
         #     true
         #     false
         xsv_permissive_cols = true

         # Required field for locatableXSV files.
         # The 0-based index of the column containing the contig for each row
         contig_column = 1

         # Required field for locatableXSV files.
         # The 0-based index of the column containing the start position for each row
         start_column = 2

         # Required field for locatableXSV files.
         # The 0-based index of the column containing the end position for each row
         end_column = 3
     

Inputs

  • A reference genome sequence.
  • The version of the reference genome sequence being used (either hg19 or hg38).
  • A VCF of variant calls to annotate.
  • The path to a folder of data sources formatted for use by Funcotator.

Output

  • A VCF containing all variants from the input file with added annotation columns corresponding to annotations from each data source that matched a given variant according to that data source's matching criteria.

Usage example

   ./gatk Funcotator \
   -R reference.fasta \
   -V input.vcf \
   -O output.vcf \
   --data-sources-path dataSourcesFolder/ \
   --ref-version hg19
 

Notes

  • This is a beta tool, and as such may generate errors or warnings.
  • This tool is the GATK analog of Oncotator.

Known Issues

A complete list of known open issues can be found on the GATK github entry for funcotator here.

Notable Issues as of 2018 Jan 3

  • Only supports VCF for inputs and outputs (Issue 3922).
  • Only supports a single GENCODE data source (Issue 3956).
  • Non-GENCODE annotations on multiallelic variants are only rendered properly for the last allele (Issue 3896).
  • The "other transcripts" annotation is missing for IGR variants (Issue 3849).
  • Only the "Format" field of input VCF files is preserved in the output VCF file (Issue 3895).
  • Codon Change and Protein Change for indels spanning splice sites are not properly rendered (Issue 3749).
  • Does not support Structural Variants (Issue 4083).

Funcotator specific arguments

This table summarizes the command-line arguments that are specific to this tool. For more details on each argument, see the list further down below the table or click on an argument name to jump directly to that entry in the list.

Argument name(s) Default value Summary
Required Arguments
--data-sources-path
[] The path to a data source folder for Funcotator. May be specified more than once to handle multiple data source folders.
--output
 -O
null Output VCF file to which annotated variants should be written.
--output-file-format
null The output file format. Either VCF or MAF. Please note that MAF output for germline use case VCFs is unsupported.
--ref-version
null The version of the Human Genome reference to use (e.g. hg19, hg38, etc.). This will correspond to a sub-folder of each data source corresponding to that data source for the given reference.
--reference
 -R
null Reference sequence file
--variant
 -V
null A VCF file containing variants
Optional Tool Arguments
--annotation-default
[] Annotations to include in all annotated variants if the annotation is not specified in the data sources (in the format :). This will add the specified annotation to every annotated variant if it is not already present.
--annotation-override
[] Override values for annotations (in the format :). Replaces existing annotations of the given name with given values.
--arguments_file
[] read one or more arguments files and add them to the command line
--cloud-index-prefetch-buffer
 -CIPB
-1 Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.
--cloud-prefetch-buffer
 -CPB
40 Size of the cloud-only prefetch buffer (in MB; 0 to disable).
--disable-bam-index-caching
 -DBIC
false If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.
--disable-sequence-dictionary-validation
false If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
--exclude-field
[] Fields that should not be rendered in the final output. Only exact name matches will be excluded.
--gcs-max-retries
 -gcs-retries
20 If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
--gcs-project-for-requester-pays
"" Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed.
--help
 -h
false display the help message
--interval-merging-rule
 -imr
ALL Interval merging rule for abutting intervals
--intervals
 -L
[] One or more genomic intervals over which to operate
--lookahead-cache-bp
100000 Number of base-pairs to cache when querying variants.
--remove-filtered-variants
false Ignore/drop variants that have been filtered in the input. These variants will not appear in the output file.
--sites-only-vcf-output
false If true, don't emit genotype fields when writing vcf file output.
--transcript-list
[] File to use as a list of transcripts (one transcript ID per line, version numbers are ignored) OR A set of transcript IDs to use for annotation to override selected transcript.
--transcript-selection-mode
CANONICAL Method of detailed transcript selection. This will select the transcript for detailed annotation (CANONICAL, ALL, or BEST_EFFECT).
--version
false display the version number for this tool
Optional Common Arguments
--add-output-sam-program-record
true If true, adds a PG tag to created SAM/BAM/CRAM files.
--add-output-vcf-command-line
true If true, adds a command line header line to created VCF files.
--create-output-bam-index
 -OBI
true If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.
--create-output-bam-md5
 -OBM
false If true, create a MD5 digest for any BAM/SAM/CRAM file created
--create-output-variant-index
 -OVI
true If true, create a VCF index when writing a coordinate-sorted VCF file.
--create-output-variant-md5
 -OVM
false If true, create a a MD5 digest any VCF file created.
--disable-read-filter
 -DF
[] Read filters to be disabled before analysis
--disable-tool-default-read-filters
false Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)
--exclude-intervals
 -XL
[] One or more genomic intervals to exclude from processing
--gatk-config-file
null A configuration file to use with the GATK.
--input
 -I
[] BAM/SAM/CRAM file containing reads
--interval-exclusion-padding
 -ixp
0 Amount of padding (in bp) to add to each interval you are excluding.
--interval-padding
 -ip
0 Amount of padding (in bp) to add to each interval you are including.
--interval-set-rule
 -isr
UNION Set merging approach to use for combining interval inputs
--lenient
 -LE
false Lenient processing of VCF files
--QUIET
false Whether to suppress job-summary info on System.err.
--read-filter
 -RF
[] Read filters to be applied before analysis
--read-index
[] Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.
--read-validation-stringency
 -VS
SILENT Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.
--seconds-between-progress-updates
10.0 Output traversal statistics every time this many seconds elapse
--sequence-dictionary
null Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--tmp-dir
null Temp directory to use.
--use-jdk-deflater
 -jdk-deflater
false Whether to use the JdkDeflater (as opposed to IntelDeflater)
--use-jdk-inflater
 -jdk-inflater
false Whether to use the JdkInflater (as opposed to IntelInflater)
--verbosity
INFO Control verbosity of logging.
Advanced Arguments
--showHidden
false display hidden arguments

Argument details

Arguments in this list are specific to this tool. Keep in mind that other arguments are available that are shared with other tools (e.g. command-line GATK arguments); see Inherited arguments above.


--add-output-sam-program-record / -add-output-sam-program-record

If true, adds a PG tag to created SAM/BAM/CRAM files.

boolean  true


--add-output-vcf-command-line / -add-output-vcf-command-line

If true, adds a command line header line to created VCF files.

boolean  true


--annotation-default / NA

Annotations to include in all annotated variants if the annotation is not specified in the data sources (in the format :). This will add the specified annotation to every annotated variant if it is not already present.

List[String]  []


--annotation-override / NA

Override values for annotations (in the format :). Replaces existing annotations of the given name with given values.

List[String]  []


--arguments_file / NA

read one or more arguments files and add them to the command line

List[File]  []


--cloud-index-prefetch-buffer / -CIPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.

int  -1  [ [ -∞  ∞ ] ]


--cloud-prefetch-buffer / -CPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable).

int  40  [ [ -∞  ∞ ] ]


--create-output-bam-index / -OBI

If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.

boolean  true


--create-output-bam-md5 / -OBM

If true, create a MD5 digest for any BAM/SAM/CRAM file created

boolean  false


--create-output-variant-index / -OVI

If true, create a VCF index when writing a coordinate-sorted VCF file.

boolean  true


--create-output-variant-md5 / -OVM

If true, create a a MD5 digest any VCF file created.

boolean  false


--data-sources-path / NA

The path to a data source folder for Funcotator. May be specified more than once to handle multiple data source folders.

R List[String]  []


--disable-bam-index-caching / -DBIC

If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.

boolean  false


--disable-read-filter / -DF

Read filters to be disabled before analysis

List[String]  []


--disable-sequence-dictionary-validation / -disable-sequence-dictionary-validation

If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!

boolean  false


--disable-tool-default-read-filters / -disable-tool-default-read-filters

Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)

boolean  false


--exclude-field / NA

Fields that should not be rendered in the final output. Only exact name matches will be excluded.

Set[String]  []


--exclude-intervals / -XL

One or more genomic intervals to exclude from processing
Use this argument to exclude certain parts of the genome from the analysis (like -L, but the opposite). This argument can be specified multiple times. You can use samtools-style intervals either explicitly on the command line (e.g. -XL 1 or -XL 1:100-200) or by loading in a file containing a list of intervals (e.g. -XL myFile.intervals).

List[String]  []


--gatk-config-file / NA

A configuration file to use with the GATK.

String  null


--gcs-max-retries / -gcs-retries

If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection

int  20  [ [ -∞  ∞ ] ]


--gcs-project-for-requester-pays / NA

Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed.

String  ""


--help / -h

display the help message

boolean  false


--input / -I

BAM/SAM/CRAM file containing reads

List[String]  []


--interval-exclusion-padding / -ixp

Amount of padding (in bp) to add to each interval you are excluding.
Use this to add padding to the intervals specified using -XL. For example, '-XL 1:100' with a padding value of 20 would turn into '-XL 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]


--interval-merging-rule / -imr

Interval merging rule for abutting intervals
By default, the program merges abutting intervals (i.e. intervals that are directly side-by-side but do not actually overlap) into a single continuous interval. However you can change this behavior if you want them to be treated as separate intervals instead.

The --interval-merging-rule argument is an enumerated type (IntervalMergingRule), which can have one of the following values:

ALL
OVERLAPPING_ONLY

IntervalMergingRule  ALL


--interval-padding / -ip

Amount of padding (in bp) to add to each interval you are including.
Use this to add padding to the intervals specified using -L. For example, '-L 1:100' with a padding value of 20 would turn into '-L 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]


--interval-set-rule / -isr

Set merging approach to use for combining interval inputs
By default, the program will take the UNION of all intervals specified using -L and/or -XL. However, you can change this setting for -L, for example if you want to take the INTERSECTION of the sets instead. E.g. to perform the analysis only on chromosome 1 exomes, you could specify -L exomes.intervals -L 1 --interval-set-rule INTERSECTION. However, it is not possible to modify the merging approach for intervals passed using -XL (they will always be merged using UNION). Note that if you specify both -L and -XL, the -XL interval set will be subtracted from the -L interval set.

The --interval-set-rule argument is an enumerated type (IntervalSetRule), which can have one of the following values:

UNION
Take the union of all intervals
INTERSECTION
Take the intersection of intervals (the subset that overlaps all intervals specified)

IntervalSetRule  UNION


--intervals / -L

One or more genomic intervals over which to operate

List[String]  []


--lenient / -LE

Lenient processing of VCF files

boolean  false


--lookahead-cache-bp / NA

Number of base-pairs to cache when querying variants.

int  100000  [ [ 0  ∞ ] ]


--output / -O

Output VCF file to which annotated variants should be written.

R File  null


--output-file-format / NA

The output file format. Either VCF or MAF. Please note that MAF output for germline use case VCFs is unsupported.

The --output-file-format argument is an enumerated type (OutputFormatType), which can have one of the following values:

VCF
MAF

R OutputFormatType  null


--QUIET / NA

Whether to suppress job-summary info on System.err.

Boolean  false


--read-filter / -RF

Read filters to be applied before analysis

List[String]  []


--read-index / -read-index

Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.

List[String]  []


--read-validation-stringency / -VS

Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.

The --read-validation-stringency argument is an enumerated type (ValidationStringency), which can have one of the following values:

STRICT
LENIENT
SILENT

ValidationStringency  SILENT


--ref-version / NA

The version of the Human Genome reference to use (e.g. hg19, hg38, etc.). This will correspond to a sub-folder of each data source corresponding to that data source for the given reference.

R String  null


--reference / -R

Reference sequence file

R String  null


--remove-filtered-variants / NA

Ignore/drop variants that have been filtered in the input. These variants will not appear in the output file.

boolean  false


--seconds-between-progress-updates / -seconds-between-progress-updates

Output traversal statistics every time this many seconds elapse

double  10.0  [ [ -∞  ∞ ] ]


--sequence-dictionary / -sequence-dictionary

Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.

String  null


--showHidden / -showHidden

display hidden arguments

boolean  false


--sites-only-vcf-output / NA

If true, don't emit genotype fields when writing vcf file output.

boolean  false


--tmp-dir / NA

Temp directory to use.

String  null


--transcript-list / NA

File to use as a list of transcripts (one transcript ID per line, version numbers are ignored) OR A set of transcript IDs to use for annotation to override selected transcript.

Set[String]  []


--transcript-selection-mode / NA

Method of detailed transcript selection. This will select the transcript for detailed annotation (CANONICAL, ALL, or BEST_EFFECT).

The --transcript-selection-mode argument is an enumerated type (TranscriptSelectionMode), which can have one of the following values:

BEST_EFFECT
BEST_EFFECT Select a transcript to be reported with details with priority on effect according to the folowing list of selection criteria: Choose the transcript that is on the custom list specified by the user. If no list was specified, treat as if no transcripts were on the list (tie). In case of tie, choose the transcript that yields the variant classification highest on the variant classification rank list (see below). If still a tie, choose the transcript with highest level of curation. Note that this means lower number is better for level (see below). If still a tie, choose the transcript with the best appris annotation (see below). If still a tie, choose the transcript with the longest transcript sequence length. If still a tie, choose the first transcript, alphabetically. Levels of Curation: 1 (verified loci) 2 manually annotated loci 3 automatically annotated loci Variant Classification Scores (See {@link org.broadinstitute.hellbender.tools.funcotator.dataSources.gencode.GencodeFuncotation.VariantClassification} as well): De_novo_Start_OutOfFrame 0 Nonsense_Mutation 0 Nonstop_Mutation 0 Missense_Mutation 1 De_novo_Start_InFrame 1 In_Frame_Del 1 In_Frame_Ins 1 Frame_Shift_Del 2 Frame_Shift_Ins 2 Frame_Shift_Sub 2 Start_Codon_SNP 3 Start_Codon_Del 3 Start_Codon_Ins 3 Start_Codon_DNP 3 Start_Codon_TNP 3 Start_Codon_ONP 3 Stop_Codon_SNP 3 Stop_Codon_Del 3 Stop_Codon_Ins 3 Stop_Codon_DNP 3 Stop_Codon_TNP 3 Stop_Codon_ONP 3 Splice_Site 4 Splice_Site_SNP 4 Splice_Site_Del 4 Splice_Site_Ins 4 Splice_Site_DNP 4 Splice_Site_TNP 4 Splice_Site_ONP 4 Splice_Site 4 miRNA 4 Silent 5 3UTR 6 5UTR 6 Intron 7 5Flank 8 3Flank 8 Non-coding_Transcript 9 IGR 20 TX-REF-MISMATCH 100 APPRIS Ranks (http://appris.bioinfo.cnio.es/): appris_principal appris_candidate_highest_score appris_candidate_longest_ccds appris_candidate_ccds appris_candidate_longest_seq appris_candidate_longest appris_candidate no appris tag present
CANONICAL
CANONICAL Select a transcript to be reported with details with priority on canonical order according to the folowing list of selection criteria: Choose the transcript that is on the custom list specified by the user. If no list was specified, treat as if all transcripts were on the list (tie). In case of tie, choose the transcript with highest level of curation. Note that this means lower number is better for level (see below). If still a tie, choose the transcript that yields the variant classification highest on the variant classification rank list (see below). If still a tie, choose the transcript with the best appris annotation (see below). If still a tie, choose the transcript with the longest transcript sequence length. If still a tie, choose the first transcript, alphabetically. Levels of Curation: 1 (verified loci) 2 manually annotated loci 3 automatically annotated loci Variant Classification Scores (See {@link GencodeFuncotation.VariantClassification} as well): De_novo_Start_OutOfFrame 0 Nonsense_Mutation 0 Nonstop_Mutation 0 Missense_Mutation 1 De_novo_Start_InFrame 1 In_Frame_Del 1 In_Frame_Ins 1 Frame_Shift_Del 2 Frame_Shift_Ins 2 Frame_Shift_Sub 2 Start_Codon_SNP 3 Start_Codon_Del 3 Start_Codon_Ins 3 Start_Codon_DNP 3 Start_Codon_TNP 3 Start_Codon_ONP 3 Stop_Codon_SNP 3 Stop_Codon_Del 3 Stop_Codon_Ins 3 Stop_Codon_DNP 3 Stop_Codon_TNP 3 Stop_Codon_ONP 3 Splice_Site 4 Splice_Site_SNP 4 Splice_Site_Del 4 Splice_Site_Ins 4 Splice_Site_DNP 4 Splice_Site_TNP 4 Splice_Site_ONP 4 Splice_Site 4 miRNA 4 Silent 5 3UTR 6 5UTR 6 Intron 7 5Flank 8 3Flank 8 Non-coding_Transcript 9 IGR 20 TX-REF-MISMATCH 100 APPRIS Ranks (http://appris.bioinfo.cnio.es/): appris_principal appris_candidate_highest_score appris_candidate_longest_ccds appris_candidate_ccds appris_candidate_longest_seq appris_candidate_longest appris_candidate no appris tag present
ALL
Same as CANONICAL, but indicates that no transcripts should be dropped. Render all overlapping transcripts.

TranscriptSelectionMode  CANONICAL


--use-jdk-deflater / -jdk-deflater

Whether to use the JdkDeflater (as opposed to IntelDeflater)

boolean  false


--use-jdk-inflater / -jdk-inflater

Whether to use the JdkInflater (as opposed to IntelInflater)

boolean  false


--variant / -V

A VCF file containing variants

R String  null


--verbosity / -verbosity

Control verbosity of logging.

The --verbosity argument is an enumerated type (LogLevel), which can have one of the following values:

ERROR
WARNING
INFO
DEBUG

LogLevel  INFO


--version / NA

display the version number for this tool

boolean  false


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GATK version 4.0.11.0 built at 19-41-2018 02:41:44.