Chart the nucleotide distribution per cycle in a SAM or BAM fileThis tool produces a chart of the nucleotide distribution per cycle in a SAM or BAM file in order to enable assessment of systematic errors at specific positions in the reads.

Interpretation notes

Increased numbers of miscalled bases will be reflected in base distribution changes and increases in the number of Ns. In general, we expect that for any given cycle, or position within reads, the relative proportions of A, T, C and G should reflect the AT:GC content of the organism's genome. Thus, for all four nucleotides, flattish lines would be expected. Deviations from this expectation, for example a spike of A at a particular cycle (position within reads), would suggest a systematic sequencing error.

Note on quality trimming

In the past, many sequencing data processing workflows included discarding the low-quality tails of reads by applying hard-clipping at some arbitrary base quality threshold value. This is no longer useful because most sophisticated analysis tools (such as the GATK variant discovery tools) are quality-aware, meaning that they are able to take base quality into account when weighing evidence provided by sequencing reads. Unnecessary clipping may interfere with other quality control evaluations and may lower the quality of analysis results. For example, trimming reduces the effectiveness of the Base Recalibration (BQSR) pre-processing step of the GATK Best Practices for Variant Discovery, which aims to correct some types of systematic biases that affect the accuracy of base quality scores.

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

Usage example:

java -jar picard.jar CollectBaseDistributionByCycle \
      CHART=collect_base_dist_by_cycle.pdf \
      I=input.bam \
      O=output.txt

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Category Diagnostics and Quality Control

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Overview

CollectBaseDistributionByCycle (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
--CHART_OUTPUT  -CHART	null	A file (with .pdf extension) to write the chart to.
--INPUT  -I	null	Input SAM or BAM file.
--OUTPUT  -O	null	The file to write the output to.
Optional Tool Arguments
--ALIGNED_READS_ONLY	false	If set to true, calculate the base distribution over aligned reads only.
--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.
--help  -h	false	display the help message
--PF_READS_ONLY	false	If set to true, calculate the base distribution over PF reads only (Illumina specific). PF reads are reads that passed the internal quality filters applied by Illumina sequencers.
--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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--ALIGNED_READS_ONLY / NA

If set to true, calculate the base distribution over aligned reads only.

boolean  false

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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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--CHART_OUTPUT / -CHART

A file (with .pdf extension) to write the chart to.

R File  null

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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

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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--OUTPUT / -O

The file to write the output to.

R File  null

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--PF_READS_ONLY / NA

If set to true, calculate the base distribution over PF reads only (Illumina specific). PF reads are reads that passed the internal quality filters applied by Illumina sequencers.

boolean  false

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