Collects metrics from reduced representation bisulfite sequencing (Rrbs) data.

This tool uses reduced representation bisulfite sequencing (Rrbs) data to determine cytosine methylation status across all reads of a genomic DNA sequence. For a primer on bisulfite sequencing and cytosine methylation, please see the corresponding GATK Dictionary entry.

Briefly, bisulfite reduction converts un-methylated cytosine (C) to uracil (U) bases. Methylated sites are not converted because they are resistant to bisulfite reduction. Subsequent to PCR amplification of the reaction products, bisulfite reduction manifests as [C -> T (+ strand) or G -> A (- strand)] base conversions. Thus, conversion rates can be calculated from the reads as follows: [CR = converted/(converted + unconverted)]. Since methylated cytosines are protected against Rrbs-mediated conversion, the methylation rate (MR) is as follows:[MR = unconverted/(converted + unconverted) = (1 - CR)].

The CpG CollectRrbsMetrics tool outputs three files including summary and detail metrics tables as well as a PDF file containing four graphs. These graphs are derived from the summary table and include a comparison of conversion rates for both CpG and non-CpG sites, the distribution of total numbers of CpG sites as a function of the CpG conversion rates, the distribution of CpG sites by the level of read coverage (depth), and the numbers of reads discarded resulting from either exceeding the mismatch rate or size (too short). The detailed metrics table includes the coordinates of all of the CpG sites for the experiment as well as the conversion rates observed for each site.

Usage example:

java -jar picard.jar CollectRrbsMetrics \
      R=reference_sequence.fasta \
      I=input.bam \
      M=basename_for_metrics_files

Please see the CollectRrbsMetrics definitions for a complete description of both the detail and summary metrics produced by this tool.

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Category Diagnostics and Quality Control

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Overview

Calculates and reports QC metrics for RRBS data based on the methylation status at individual C/G bases as well as CpG sites across all reads in the input BAM/SAM file.

CollectRrbsMetrics (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	null	The BAM or SAM file containing aligned reads. Must be coordinate sorted
--METRICS_FILE_PREFIX  -M	null	Base name for output files
--REFERENCE  -R	null	The reference sequence fasta file
Optional Tool Arguments
--arguments_file	[]	read one or more arguments files and add them to the command line
--ASSUME_SORTED  -AS	false	If true, assume that the input file is coordinate sorted even if the header says otherwise.
--C_QUALITY_THRESHOLD	20	Threshold for base quality of a C base before it is considered
--help  -h	false	display the help message
--MAX_MISMATCH_RATE	0.1	Maximum percentage of mismatches in a read for it to be considered, with a range of 0-1
--METRIC_ACCUMULATION_LEVEL  -LEVEL	[ALL_READS]	The level(s) at which to accumulate metrics.
--MINIMUM_READ_LENGTH	5	Minimum read length
--NEXT_BASE_QUALITY_THRESHOLD	10	Threshold for quality of a base next to a C before the C base is considered
--SEQUENCE_NAMES	[]	Set of sequence names to consider, if not specified all sequences will be used
--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.
--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 / NA

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

List[File]  []

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--ASSUME_SORTED / -AS

If true, assume that the input file is coordinate sorted even if the header says otherwise.

boolean  false

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--C_QUALITY_THRESHOLD / NA

Threshold for base quality of a C base before it is considered

int  20  [ [ -∞  ∞ ] ]

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--COMPRESSION_LEVEL / NA

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

int  5  [ [ -∞  ∞ ] ]

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--CREATE_INDEX / NA

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

Boolean  false

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--CREATE_MD5_FILE / NA

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

boolean  false

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--GA4GH_CLIENT_SECRETS / NA

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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--INPUT / -I

The BAM or SAM file containing aligned reads. Must be coordinate sorted

R File  null

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--MAX_MISMATCH_RATE / NA

Maximum percentage of mismatches in a read for it to be considered, with a range of 0-1

double  0.1  [ [ -∞  ∞ ] ]

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--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  [ [ -∞  ∞ ] ]

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--METRIC_ACCUMULATION_LEVEL / -LEVEL

The level(s) at which to accumulate metrics.

Set[MetricAccumulationLevel]  [ALL_READS]

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--METRICS_FILE_PREFIX / -M

Base name for output files

R String  null

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--MINIMUM_READ_LENGTH / NA

Minimum read length

int  5  [ [ -∞  ∞ ] ]

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--NEXT_BASE_QUALITY_THRESHOLD / NA

Threshold for quality of a base next to a C before the C base is considered

int  10  [ [ -∞  ∞ ] ]

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--QUIET / NA

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

Boolean  false

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--REFERENCE / -R

The reference sequence fasta file

R File  null

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--SEQUENCE_NAMES / NA

Set of sequence names to consider, if not specified all sequences will be used

Set[String]  []

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--showHidden / -showHidden

display hidden arguments

boolean  false

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--TMP_DIR / NA

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 / 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

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--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

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--version / NA

display the version number for this tool

boolean  false

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GATK version 4.1.6.0-SNAPSHOT built at Thu, 2 Apr 2020 14:54:17 -0400.