This tool provides useful metrics for validating library construction including the insert size distribution and read orientation of paired-end libraries.

The expected proportions of these metrics vary depending on the type of library preparation used, resulting from technical differences between pair-end libraries and mate-pair libraries. For a brief primer on paired-end sequencing and mate-pair reads, see the GATK Dictionary.

The CollectInsertSizeMetrics tool outputs the percentages of read pairs in each of the three orientations (FR, RF, and TANDEM) as a histogram. In addition, the insert size distribution is output as both a histogram (.insert_size_Histogram.pdf) and as a data table (.insert_size_metrics.txt).

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

Usage example:

java -jar picard.jar CollectInsertSizeMetrics \
      I=input.bam \
      O=insert_size_metrics.txt \
      H=insert_size_histogram.pdf \
      M=0.5

Note: If processing a small file, set the minimum percentage option (M) to 0.5, otherwise an error may occur.

Please see InsertSizeMetrics for detailed explanations of each metric.

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Collect metrics about the insert size distribution of a paired-end library.

Category Diagnostics and Quality Control

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Overview

Command line program to read non-duplicate insert sizes, create a Histogram and report distribution statistics.

CollectInsertSizeMetrics (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
--Histogram_FILE  -H	null	File to write insert size Histogram chart to.
--INPUT  -I	null	Input SAM or BAM file.
--OUTPUT  -O	null	File to write the output to.
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.
--DEVIATIONS	10.0	Generate mean, sd and plots by trimming the data down to MEDIAN + DEVIATIONS*MEDIAN_ABSOLUTE_DEVIATION. This is done because insert size data typically includes enough anomalous values from chimeras and other artifacts to make the mean and sd grossly misleading regarding the real distribution.
--help  -h	false	display the help message
--HISTOGRAM_WIDTH  -W	null	Explicitly sets the Histogram width, overriding automatic truncation of Histogram tail. Also, when calculating mean and standard deviation, only bins <= Histogram_WIDTH will be included.
--INCLUDE_DUPLICATES	false	If true, also include reads marked as duplicates in the insert size histogram.
--METRIC_ACCUMULATION_LEVEL  -LEVEL	[ALL_READS]	The level(s) at which to accumulate metrics.
--MINIMUM_PCT  -M	0.05	When generating the Histogram, discard any data categories (out of FR, TANDEM, RF) that have fewer than this percentage of overall reads. (Range: 0 to 1).
--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.

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

boolean  true

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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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--DEVIATIONS / NA

Generate mean, sd and plots by trimming the data down to MEDIAN + DEVIATIONS*MEDIAN_ABSOLUTE_DEVIATION. This is done because insert size data typically includes enough anomalous values from chimeras and other artifacts to make the mean and sd grossly misleading regarding the real distribution.

double  10.0  [ [ -∞  ∞ ] ]

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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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--Histogram_FILE / -H

File to write insert size Histogram chart to.

R File  null

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--HISTOGRAM_WIDTH / -W

Explicitly sets the Histogram width, overriding automatic truncation of Histogram tail. Also, when calculating mean and standard deviation, only bins <= Histogram_WIDTH will be included.

Integer  null

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--INCLUDE_DUPLICATES / NA

If true, also include reads marked as duplicates in the insert size histogram.

boolean  false

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--INPUT / -I

Input SAM or BAM file.

R File  null

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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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--MINIMUM_PCT / -M

When generating the Histogram, discard any data categories (out of FR, TANDEM, RF) that have fewer than this percentage of overall reads. (Range: 0 to 1).

float  0.05  [ [ -∞  ∞ ] ]

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--OUTPUT / -O

File to write the output to.

R File  null

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--QUIET / NA

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

Boolean  false

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--REFERENCE_SEQUENCE / -R

Reference sequence file.

File  null

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--showHidden / -showHidden

display hidden arguments

boolean  false

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--STOP_AFTER / NA

Stop after processing N reads, mainly for debugging.

long  0  [ [ -∞  ∞ ] ]

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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 4.0.5.1 built at 25-59-2019 01:59:53.