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CollectRnaSeqMetrics (Picard)

Produces RNA alignment metrics for a SAM or BAM file.

This tool takes a SAM/BAM file containing the aligned reads from an RNAseq experiment and produces metrics describing the distribution of the bases within the transcripts. It calculates the total numbers and the fractions of nucleotides within specific genomic regions including untranslated regions (UTRs), introns, intergenic sequences (between discrete genes), and peptide-coding sequences (exons). This tool also determines the numbers of bases that pass quality filters that are specific to Illumina data (PF_BASES). For more information please see the corresponding GATK Dictionary entry.

Other metrics include the median coverage (depth), the ratios of 5 prime /3 prime-biases, and the numbers of reads with the correct/incorrect strand designation. The 5 prime /3 prime-bias results from errors introduced by reverse transcriptase enzymes during library construction, ultimately leading to the over-representation of either the 5 prime or 3 prime ends of transcripts. Please see the CollectRnaSeqMetrics definitions for details on how these biases are calculated.

The sequence input must be a valid SAM/BAM file containing RNAseq data aligned by an RNAseq-aware genome aligner such a STAR or TopHat. The tool also requires a REF_FLAT file, a tab-delimited file containing information about the location of RNA transcripts, exon start and stop sites, etc. For an example refFlat file for GRCh38, see refFlat.txt.gz at http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database. The first five lines of the tab-limited text file appear as follows.

DDX11L1	NR_046018	chr1	+	11873	14409	14409	14409	3	11873,12612,13220,	12227,12721,14409,WASH7P	NR_024540	chr1	-	14361	29370	29370	29370	11	14361,14969,15795,16606,16857,17232,17605,17914,18267,24737,29320,	14829,15038,15947,16765,17055,17368,17742,18061,18366,24891,29370,DLGAP2-AS1	NR_103863	chr8_KI270926v1_alt	-	33083	35050	35050	35050	3	33083,33761,35028,	33281,33899,35050,MIR570	NR_030296	chr3	+	195699400	195699497	195699497	195699497	1	195699400,	195699497,MIR548A3	NR_030330	chr8	-	104484368	104484465	104484465	104484465	1	104484368,	104484465,

Note: Metrics labeled as percentages are actually expressed as fractions!

Usage example:

java -jar picard.jar CollectRnaSeqMetrics \
I=input.bam \
O=output.RNA_Metrics \
REF_FLAT=ref_flat.txt \
STRAND=SECOND_READ_TRANSCRIPTION_STRAND \
RIBOSOMAL_INTERVALS=ribosomal.interval_list
Please see the CollectRnaSeqMetrics definitions for a complete description of the metrics produced by this tool.

Category Diagnostics and Quality Control


Overview

CollectRnaSeqMetrics (Picard) 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
--IGNORE_SEQUENCE
[] If a read maps to a sequence specified with this option, all the bases in the read are counted as ignored bases. These reads are not counted as
--INPUT
 -I
null Input SAM or BAM file.
--OUTPUT
 -O
null File to write the output to.
--REF_FLAT
null Gene annotations in refFlat form. Format described here: http://genome.ucsc.edu/goldenPath/gbdDescriptionsOld.html#RefFlat
--STRAND_SPECIFICITY
 -STRAND
null For strand-specific library prep. For unpaired reads, use FIRST_READ_TRANSCRIPTION_STRAND if the reads are expected to be on the transcription strand.
Optional Tool Arguments
--arguments_file
[] read one or more arguments files and add them to the command line
--ASSUME_SORTED
 -AS
true If true (default), then the sort order in the header file will be ignored.
--CHART_OUTPUT
 -CHART
null The PDF file to write out a plot of normalized position vs. coverage.
--help
 -h
false display the help message
--METRIC_ACCUMULATION_LEVEL
 -LEVEL
[ALL_READS] The level(s) at which to accumulate metrics.
--MINIMUM_LENGTH
500 When calculating coverage based values (e.g. CV of coverage) only use transcripts of this length or greater.
--RIBOSOMAL_INTERVALS
null Location of rRNA sequences in genome, in interval_list format. If not specified no bases will be identified as being ribosomal. Format described here:
--RRNA_FRAGMENT_PERCENTAGE
0.8 This percentage of the length of a fragment must overlap one of the ribosomal intervals for a read or read pair to be considered rRNA.
--STOP_AFTER
0 Stop after processing N reads, mainly for debugging.
--version
false display the version number for this tool
Optional Common Arguments
--COMPRESSION_LEVEL
5 Compression level for all compressed files created (e.g. BAM and VCF).
--CREATE_INDEX
false Whether to create a BAM index when writing a coordinate-sorted BAM file.
--CREATE_MD5_FILE
false Whether to create an MD5 digest for any BAM or FASTQ files created.
--GA4GH_CLIENT_SECRETS
client_secrets.json Google Genomics API client_secrets.json file path.
--MAX_RECORDS_IN_RAM
500000 When writing files that need to be sorted, this will specify the number of records stored in RAM before spilling to disk. Increasing this number reduces the number of file handles needed to sort the file, and increases the amount of RAM needed.
--QUIET
false Whether to suppress job-summary info on System.err.
--REFERENCE_SEQUENCE
 -R
null Reference sequence file.
--TMP_DIR
[] One or more directories with space available to be used by this program for temporary storage of working files
--USE_JDK_DEFLATER
 -use_jdk_deflater
false Use the JDK Deflater instead of the Intel Deflater for writing compressed output
--USE_JDK_INFLATER
 -use_jdk_inflater
false Use the JDK Inflater instead of the Intel Inflater for reading compressed input
--VALIDATION_STRINGENCY
STRICT Validation stringency for all SAM files read by this program. Setting stringency to SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.
--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.


--arguments_file / NA

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

List[File]  []


--ASSUME_SORTED / -AS

If true (default), then the sort order in the header file will be ignored.

boolean  true


--CHART_OUTPUT / -CHART

The PDF file to write out a plot of normalized position vs. coverage.

File  null


--COMPRESSION_LEVEL / NA

Compression level for all compressed files created (e.g. BAM and VCF).

int  5  [ [ -∞  ∞ ] ]


--CREATE_INDEX / NA

Whether to create a BAM index when writing a coordinate-sorted BAM file.

Boolean  false


--CREATE_MD5_FILE / NA

Whether to create an MD5 digest for any BAM or FASTQ files created.

boolean  false


--GA4GH_CLIENT_SECRETS / NA

Google Genomics API client_secrets.json file path.

String  client_secrets.json


--help / -h

display the help message

boolean  false


--IGNORE_SEQUENCE / NA

If a read maps to a sequence specified with this option, all the bases in the read are counted as ignored bases. These reads are not counted as

R Set[String]  []


--INPUT / -I

Input SAM or BAM file.

R File  null


--MAX_RECORDS_IN_RAM / NA

When writing files that need to be sorted, this will specify the number of records stored in RAM before spilling to disk. Increasing this number reduces the number of file handles needed to sort the file, and increases the amount of RAM needed.

Integer  500000  [ [ -∞  ∞ ] ]


--METRIC_ACCUMULATION_LEVEL / -LEVEL

The level(s) at which to accumulate metrics.

Set[MetricAccumulationLevel]  [ALL_READS]


--MINIMUM_LENGTH / NA

When calculating coverage based values (e.g. CV of coverage) only use transcripts of this length or greater.

int  500  [ [ -∞  ∞ ] ]


--OUTPUT / -O

File to write the output to.

R File  null


--QUIET / NA

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

Boolean  false


--REF_FLAT / NA

Gene annotations in refFlat form. Format described here: http://genome.ucsc.edu/goldenPath/gbdDescriptionsOld.html#RefFlat

R File  null


--REFERENCE_SEQUENCE / -R

Reference sequence file.

File  null


--RIBOSOMAL_INTERVALS / NA

Location of rRNA sequences in genome, in interval_list format. If not specified no bases will be identified as being ribosomal. Format described here:

File  null


--RRNA_FRAGMENT_PERCENTAGE / NA

This percentage of the length of a fragment must overlap one of the ribosomal intervals for a read or read pair to be considered rRNA.

double  0.8  [ [ -∞  ∞ ] ]


--showHidden / -showHidden

display hidden arguments

boolean  false


--STOP_AFTER / NA

Stop after processing N reads, mainly for debugging.

long  0  [ [ -∞  ∞ ] ]


--STRAND_SPECIFICITY / -STRAND

For strand-specific library prep. For unpaired reads, use FIRST_READ_TRANSCRIPTION_STRAND if the reads are expected to be on the transcription strand.

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

NONE
FIRST_READ_TRANSCRIPTION_STRAND
SECOND_READ_TRANSCRIPTION_STRAND

R StrandSpecificity  null


--TMP_DIR / NA

One or more directories with space available to be used by this program for temporary storage of working files

List[File]  []


--USE_JDK_DEFLATER / -use_jdk_deflater

Use the JDK Deflater instead of the Intel Deflater for writing compressed output

Boolean  false


--USE_JDK_INFLATER / -use_jdk_inflater

Use the JDK Inflater instead of the Intel Inflater for reading compressed input

Boolean  false


--VALIDATION_STRINGENCY / NA

Validation stringency for all SAM files read by this program. Setting stringency to 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 --VALIDATION_STRINGENCY argument is an enumerated type (ValidationStringency), which can have one of the following values:

STRICT
LENIENT
SILENT

ValidationStringency  STRICT


--VERBOSITY / NA

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 4.1.0.0 built at Wed, 30 Jan 2019 10:21:04 +0530.