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.

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Category Diagnostics and Quality Control

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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
--INPUT  -I		Input SAM or BAM file.
--OUTPUT  -O		The file to write the output to.
--REF_FLAT		Gene annotations in refFlat form. Format described here: http://genome.ucsc.edu/goldenPath/gbdDescriptionsOld.html#RefFlat
--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.
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		The PDF file to write out a plot of normalized position vs. coverage.
--END_BIAS_BASES	100	The distance into a transcript over which 5' and 3' bias is calculated.
--help  -h	false	display the help message
--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 towards any metrics, except for the PF_BASES field.
--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		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 an index when writing VCF or coordinate sorted BAM output.
--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		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.

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--arguments_file

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

List[File]  []

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--ASSUME_SORTED / -AS

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

boolean  true

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--CHART_OUTPUT / -CHART

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

File  null

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--COMPRESSION_LEVEL

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

int  5  [ [ -∞  ∞ ] ]

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--CREATE_INDEX

Whether to create an index when writing VCF or coordinate sorted BAM output.

Boolean  false

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--CREATE_MD5_FILE

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

boolean  false

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--END_BIAS_BASES

The distance into a transcript over which 5' and 3' bias is calculated.

int  100  [ [ -∞  ∞ ] ]

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--GA4GH_CLIENT_SECRETS

Google Genomics API client_secrets.json file path.

String  client_secrets.json

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--help / -h

display the help message

boolean  false

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--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 towards any metrics, except for the PF_BASES field.

Set[String]  []

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--INPUT / -I

Input SAM or BAM file.

R File  null

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--MAX_RECORDS_IN_RAM

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  [ [ -∞  ∞ ] ]

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--METRIC_ACCUMULATION_LEVEL / -LEVEL

The level(s) at which to accumulate metrics.

The --METRIC_ACCUMULATION_LEVEL argument is an enumerated type (Set[MetricAccumulationLevel]), which can have one of the following values:

ALL_READS

SAMPLE

LIBRARY

READ_GROUP

Set[MetricAccumulationLevel]  [ALL_READS]

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--MINIMUM_LENGTH

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

int  500  [ [ -∞  ∞ ] ]

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--OUTPUT / -O

The file to write the output to.

R File  null

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--QUIET

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

Boolean  false

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--REF_FLAT

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

R File  null

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--REFERENCE_SEQUENCE / -R

Reference sequence file.

File  null

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--RIBOSOMAL_INTERVALS

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

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--RRNA_FRAGMENT_PERCENTAGE

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  [ [ -∞  ∞ ] ]

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--showHidden / -showHidden

display hidden arguments

boolean  false

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--STOP_AFTER

Stop after processing N reads, mainly for debugging.

long  0  [ [ -∞  ∞ ] ]

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--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

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--TMP_DIR

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

List[File]  []

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--USE_JDK_DEFLATER / -use_jdk_deflater

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

Boolean  false

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--USE_JDK_INFLATER / -use_jdk_inflater

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

Boolean  false

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--VALIDATION_STRINGENCY

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

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--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

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--version

display the version number for this tool

boolean  false

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GATK version 4.2.2.0-SNAPSHOT built at Thu, 19 Aug 2021 09:49:28 -0700.