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!
java -jar picard.jar CollectRnaSeqMetrics \Please see the CollectRnaSeqMetrics definitions for a complete description of the metrics produced by this tool.
I=input.bam \
O=output.RNA_Metrics \
REF_FLAT=ref_flat.txt \
STRAND=SECOND_READ_TRANSCRIPTION_STRAND \
RIBOSOMAL_INTERVALS=ribosomal.interval_list
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/BAM/CRAM 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. | |
| --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 | |
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.
read one or more arguments files and add them to the command line
List[File] []
If true (default), then the sort order in the header file will be ignored.
boolean true
The PDF file to write out a plot of normalized position vs. coverage.
File null
Compression level for all compressed files created (e.g. BAM and VCF).
int 5 [ [ -∞ ∞ ] ]
Whether to create an index when writing VCF or coordinate sorted BAM output.
Boolean false
Whether to create an MD5 digest for any BAM or FASTQ files created.
boolean false
The distance into a transcript over which 5' and 3' bias is calculated.
int 100 [ [ -∞ ∞ ] ]
display the help message
boolean false
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] []
Input SAM/BAM/CRAM file.
R File null
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 [ [ -∞ ∞ ] ]
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:
Set[MetricAccumulationLevel] [ALL_READS]
When calculating coverage based values (e.g. CV of coverage) only use transcripts of this length or greater.
int 500 [ [ -∞ ∞ ] ]
The file to write the output to.
R File null
Whether to suppress job-summary info on System.err.
Boolean false
Gene annotations in refFlat form. Format described here: http://genome.ucsc.edu/goldenPath/gbdDescriptionsOld.html#RefFlat
R File null
Reference sequence file.
PicardHtsPath 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:
File null
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 [ [ -∞ ∞ ] ]
display hidden arguments
boolean false
Stop after processing N reads, mainly for debugging.
long 0 [ [ -∞ ∞ ] ]
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:
R StrandSpecificity null
One or more directories with space available to be used by this program for temporary storage of working files
List[File] []
Use the JDK Deflater instead of the Intel Deflater for writing compressed output
Boolean false
Use the JDK Inflater instead of the Intel Inflater for reading compressed input
Boolean false
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:
ValidationStringency STRICT
Control verbosity of logging.
The --VERBOSITY argument is an enumerated type (LogLevel), which can have one of the following values:
LogLevel INFO
display the version number for this tool
boolean false
See also General Documentation | Tool Docs Index Tool Documentation Index | Support Forum
GATK version 4.6.2.0 built at Sun, 13 Apr 2025 13:21:43 -0400.