Generate coverage summary information for reads data

Category Coverage Analysis

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Overview

Assess sequence coverage by a wide array of metrics, partitioned by sample, read group, or library

This tool processes a set of bam files to determine coverage at different levels of partitioning and aggregation. Coverage can be analyzed per locus, per interval, per gene, or in total; can be partitioned by sample, by read group, by technology, by center, or by library; and can be summarized by mean, median, quartiles, and/or percentage of bases covered to or beyond a threshold. Additionally, reads and bases can be filtered by mapping or base quality score.

Input

• One or more bam files (with proper headers) to be analyzed for coverage statistics
• (Optional) A REFSEQ file to aggregate coverage to the gene level (for information about creating the REFSEQ file, please consult the online documentation)

Output

Tables pertaining to different coverage summaries. Suffix on the table files declares the contents:

• no suffix: per locus coverage
• _summary: total, mean, median, quartiles, and threshold proportions, aggregated over all bases
• _statistics: coverage histograms (# locus with X coverage), aggregated over all bases
• _interval_summary: total, mean, median, quartiles, and threshold proportions, aggregated per interval
• _interval_statistics: 2x2 table of # of intervals covered to >= X depth in >=Y samples
• _gene_summary: total, mean, median, quartiles, and threshold proportions, aggregated per gene
• _gene_statistics: 2x2 table of # of genes covered to >= X depth in >= Y samples. In its current incarnation it will not include genes not at least partially covered (see --omit-genes-not-entirely-covered-by-traversal for details)
• _cumulative_coverage_counts: coverage histograms (# locus with >= X coverage), aggregated over all bases
• _cumulative_coverage_proportions: proprotions of loci with >= X coverage, aggregated over all bases

Notes

• DepthOfCoverage currently only supports typical nucleotide (and N) bases, IUPAC ambiguity codes or other non-ATCGN bases will cause exceptions
• Read filters are applied to the reads before being counted in coverage information. By default Duplicate Marked and non-primary alignments are not counted. This can be disabled with --disable-tool-default-read-filters.
• In order to filter reads out by their mapping qualities, the recommended approach is to use the MappingQualityReadFilter with the --minimum-mapping-quality or --maximum-mapping-quality arguments specified

Usage example

 gatk \
   DepthOfCoverage \
   -R reference.fasta \
   -O file_name_base \
   -I input_bams.list
   [-gene-list refSeq.sorted.refseq] \
   [-partition-type readgroup] \
   [--summary-coverage-threshold 4 --summary-coverage-threshold 6 --summary-coverage-threshold 10] \
   [-L my_capture_genes.interval_list]
 

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Additional Information

Read filters

These Read Filters are automatically applied to the data by the Engine before processing by DepthOfCoverage.

• NotSecondaryAlignmentReadFilter
• MappedReadFilter
• NotDuplicateReadFilter
• WellformedReadFilter

DepthOfCoverage 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		BAM/SAM/CRAM file containing reads
--intervals  -L		One or more genomic intervals over which to operate
--output  -O		Base file location to which to write coverage summary information, must be a path to a file
--reference  -R		Reference sequence file
Optional Tool Arguments
--arguments_file		read one or more arguments files and add them to the command line
--calculate-coverage-over-genes  -gene-list		Calculate coverage statistics over this list of genes
--cloud-index-prefetch-buffer  -CIPB	-1	Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.
--cloud-prefetch-buffer  -CPB	40	Size of the cloud-only prefetch buffer (in MB; 0 to disable).
--count-type	COUNT_READS	How should overlapping reads from the same fragment be handled? NOTE: currently only COUNT_READS is supported
--disable-bam-index-caching  -DBIC	false	If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.
--disable-sequence-dictionary-validation	false	If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
--gcs-max-retries  -gcs-retries	20	If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
--gcs-project-for-requester-pays		Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed. User must have storage.buckets.get permission on the bucket being accessed.
--help  -h	false	display the help message
--interval-merging-rule  -imr	ALL	Interval merging rule for abutting intervals
--max-base-quality	127	Maximum quality of bases to count towards depth
--max-depth-per-sample	0	Maximum number of reads to retain per sample per locus. Reads above this threshold will be downsampled. Set to 0 to disable.
--min-base-quality	0	Minimum quality of bases to count towards depth
--omit-depth-output-at-each-base	false	Do not output depth of coverage at each base
--omit-genes-not-entirely-covered-by-traversal	false	Do not output gene summary if it was not completely covered by traversal intervals
--omit-interval-statistics	false	Do not calculate per-interval statistics
--omit-locus-table	false	Do not calculate per-sample per-depth counts of loci
--omit-per-sample-statistics	false	Do not output the summary files per-sample
--output-format	CSV	The format of the output file
--partition-type  -pt	[sample]	Partition type for depth of coverage
--print-base-counts	false	Add base counts to per-locus output
--sites-only-vcf-output	false	If true, don't emit genotype fields when writing vcf file output.
--version	false	display the version number for this tool
Optional Common Arguments
--add-output-sam-program-record	true	If true, adds a PG tag to created SAM/BAM/CRAM files.
--add-output-vcf-command-line	true	If true, adds a command line header line to created VCF files.
--create-output-bam-index  -OBI	true	If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.
--create-output-bam-md5  -OBM	false	If true, create a MD5 digest for any BAM/SAM/CRAM file created
--create-output-variant-index  -OVI	true	If true, create a VCF index when writing a coordinate-sorted VCF file.
--create-output-variant-md5  -OVM	false	If true, create a a MD5 digest any VCF file created.
--disable-read-filter  -DF		Read filters to be disabled before analysis
--disable-tool-default-read-filters	false	Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)
--exclude-intervals  -XL		One or more genomic intervals to exclude from processing
--gatk-config-file		A configuration file to use with the GATK.
--interval-exclusion-padding  -ixp	0	Amount of padding (in bp) to add to each interval you are excluding.
--interval-padding  -ip	0	Amount of padding (in bp) to add to each interval you are including.
--interval-set-rule  -isr	UNION	Set merging approach to use for combining interval inputs
--lenient  -LE	false	Lenient processing of VCF files
--max-variants-per-shard	0	If non-zero, partitions VCF output into shards, each containing up to the given number of records.
--QUIET	false	Whether to suppress job-summary info on System.err.
--read-filter  -RF		Read filters to be applied before analysis
--read-index		Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.
--read-validation-stringency  -VS	SILENT	Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.
--seconds-between-progress-updates	10.0	Output traversal statistics every time this many seconds elapse
--sequence-dictionary		Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--tmp-dir		Temp directory to use.
--use-jdk-deflater  -jdk-deflater	false	Whether to use the JdkDeflater (as opposed to IntelDeflater)
--use-jdk-inflater  -jdk-inflater	false	Whether to use the JdkInflater (as opposed to IntelInflater)
--verbosity	INFO	Control verbosity of logging.
Advanced Arguments
--ignore-deletion-sites	false	Ignore sites consisting only of deletions
--include-deletions	false	Include information on deletions alongside other bases in output table counts
--include-ref-n-sites	false	Include sites where the reference is N
--nBins	499	Number of bins to use for granular binning
--showHidden	false	display hidden arguments
--start	1	Starting (left endpoint) for granular binning
--stop	500	Ending (right endpoint) for granular binning
--summary-coverage-threshold	[15]	Coverage threshold (in percent) for summarizing statistics

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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--add-output-sam-program-record / -add-output-sam-program-record

If true, adds a PG tag to created SAM/BAM/CRAM files.

boolean  true

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--add-output-vcf-command-line / -add-output-vcf-command-line

If true, adds a command line header line to created VCF files.

boolean  true

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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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--calculate-coverage-over-genes / -gene-list

Calculate coverage statistics over this list of genes
Specify a RefSeq file for use in aggregating coverage statistics over genes.

This argument is incompatible with --omit-interval-statistics. A warning will be logged and no output file will be produced for the gene list if these arguments are enabled together.

Exclusion: This argument cannot be used at the same time as omit-interval-statistics.

List[String]  []

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--cloud-index-prefetch-buffer / -CIPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.

int  -1  [ [ -∞  ∞ ] ]

---

--cloud-prefetch-buffer / -CPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable).

int  40  [ [ -∞  ∞ ] ]

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--count-type

How should overlapping reads from the same fragment be handled? NOTE: currently only COUNT_READS is supported

The --count-type argument is an enumerated type (CountPileupType), which can have one of the following values:

COUNT_READS

Count all reads independently (even if from the same fragment).

COUNT_FRAGMENTS

Count all fragments (even if the reads that compose the fragment are not consistent at that base).

COUNT_FRAGMENTS_REQUIRE_SAME_BASE

Count all fragments (but only if the reads that compose the fragment are consistent at that base).

CountPileupType  COUNT_READS

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--create-output-bam-index / -OBI

If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.

boolean  true

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--create-output-bam-md5 / -OBM

If true, create a MD5 digest for any BAM/SAM/CRAM file created

boolean  false

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--create-output-variant-index / -OVI

If true, create a VCF index when writing a coordinate-sorted VCF file.

boolean  true

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--create-output-variant-md5 / -OVM

If true, create a a MD5 digest any VCF file created.

boolean  false

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--disable-bam-index-caching / -DBIC

If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.

boolean  false

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--disable-read-filter / -DF

Read filters to be disabled before analysis

List[String]  []

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--disable-sequence-dictionary-validation / -disable-sequence-dictionary-validation

If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!

boolean  false

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--disable-tool-default-read-filters / -disable-tool-default-read-filters

Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)

boolean  false

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--exclude-intervals / -XL

One or more genomic intervals to exclude from processing
Use this argument to exclude certain parts of the genome from the analysis (like -L, but the opposite). This argument can be specified multiple times. You can use samtools-style intervals either explicitly on the command line (e.g. -XL 1 or -XL 1:100-200) or by loading in a file containing a list of intervals (e.g. -XL myFile.intervals).

List[String]  []

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--gatk-config-file

A configuration file to use with the GATK.

String  null

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--gcs-max-retries / -gcs-retries

If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection

int  20  [ [ -∞  ∞ ] ]

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--gcs-project-for-requester-pays

Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed. User must have storage.buckets.get permission on the bucket being accessed.

String  ""

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--help / -h

display the help message

boolean  false

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--ignore-deletion-sites

Ignore sites consisting only of deletions

boolean  false

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--include-deletions

Include information on deletions alongside other bases in output table counts
Consider a spanning deletion as contributing to coverage. Also enables deletion counts in per-base output.

boolean  false

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--include-ref-n-sites

Include sites where the reference is N
Normally, sites where the reference is N (or another non-canonical base) are skipped. If this option is enabled, these sites will be included in DoC calculations if there is coverage from neighboring reads.

boolean  false

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--input / -I

BAM/SAM/CRAM file containing reads

R List[GATKPath]  []

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--interval-exclusion-padding / -ixp

Amount of padding (in bp) to add to each interval you are excluding.
Use this to add padding to the intervals specified using -XL. For example, '-XL 1:100' with a padding value of 20 would turn into '-XL 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]

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--interval-merging-rule / -imr

Interval merging rule for abutting intervals
By default, the program merges abutting intervals (i.e. intervals that are directly side-by-side but do not actually overlap) into a single continuous interval. However you can change this behavior if you want them to be treated as separate intervals instead.

The --interval-merging-rule argument is an enumerated type (IntervalMergingRule), which can have one of the following values:

ALL

OVERLAPPING_ONLY

IntervalMergingRule  ALL

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--interval-padding / -ip

Amount of padding (in bp) to add to each interval you are including.
Use this to add padding to the intervals specified using -L. For example, '-L 1:100' with a padding value of 20 would turn into '-L 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]

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--interval-set-rule / -isr

Set merging approach to use for combining interval inputs
By default, the program will take the UNION of all intervals specified using -L and/or -XL. However, you can change this setting for -L, for example if you want to take the INTERSECTION of the sets instead. E.g. to perform the analysis only on chromosome 1 exomes, you could specify -L exomes.intervals -L 1 --interval-set-rule INTERSECTION. However, it is not possible to modify the merging approach for intervals passed using -XL (they will always be merged using UNION). Note that if you specify both -L and -XL, the -XL interval set will be subtracted from the -L interval set.

The --interval-set-rule argument is an enumerated type (IntervalSetRule), which can have one of the following values:

UNION

Take the union of all intervals

INTERSECTION

Take the intersection of intervals (the subset that overlaps all intervals specified)

IntervalSetRule  UNION

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--intervals / -L

One or more genomic intervals over which to operate

R List[String]  []

---

--lenient / -LE

Lenient processing of VCF files

boolean  false

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--max-base-quality

Maximum quality of bases to count towards depth
Bases with quality scores higher than this threshold will be skipped. The default value is the largest number that can be represented as a byte.

byte  127  [ [ 0  127 ] ]

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--max-depth-per-sample / -max-depth-per-sample

Maximum number of reads to retain per sample per locus. Reads above this threshold will be downsampled. Set to 0 to disable.

int  0  [ [ -∞  ∞ ] ]

---

--max-variants-per-shard

If non-zero, partitions VCF output into shards, each containing up to the given number of records.

int  0  [ [ 0  ∞ ] ]

---

--min-base-quality

Minimum quality of bases to count towards depth
Bases with quality scores lower than this threshold will be skipped. This is set to -1 by default to disable the evaluation and ignore this threshold.

byte  0  [ [ 0  127 ] ]

---

--nBins

Number of bins to use for granular binning
Sets the number of bins for granular binning

int  499  [ [ 0  [ 1  ∞ ] ]

---

--omit-depth-output-at-each-base

Do not output depth of coverage at each base
Disabling the tabulation of total coverage at every base should speed up processing.

boolean  false

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--omit-genes-not-entirely-covered-by-traversal

Do not output gene summary if it was not completely covered by traversal intervals
Remove genes from the gene summary output file if all of its exon bases were not completely covered by traversal.

boolean  false

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--omit-interval-statistics

Do not calculate per-interval statistics
Disabling the tabulation of interval statistics (mean, median, quartiles AND # intervals by sample by coverage) should speed up processing.

Exclusion: This argument cannot be used at the same time as calculate-coverage-over-genes.

boolean  false

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--omit-locus-table

Do not calculate per-sample per-depth counts of loci
Disabling the tabulation of locus statistics (# loci covered by sample by coverage) should speed up processing.

boolean  false

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--omit-per-sample-statistics

Do not output the summary files per-sample
This option simply disables writing separate files for per-sample summary statistics (total, mean, median, quartile coverage per sample). These statistics are still calculated internally, so enabling this option will not improve runtime.

boolean  false

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--output / -O

Base file location to which to write coverage summary information, must be a path to a file
Base file name about which to create the coverage information

R String  null

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--output-format

The format of the output file
Output file format (e.g. csv, table, rtable); defaults to r-readable table.

The --output-format argument is an enumerated type (DEPTH_OF_COVERAGE_OUTPUT_FORMAT), which can have one of the following values:

TABLE

CSV

DEPTH_OF_COVERAGE_OUTPUT_FORMAT  CSV

---

--partition-type / -pt

Partition type for depth of coverage
By default, coverage is partitioned by sample, but it can be any combination of sample, readgroup and/or library.

The --partition-type argument is an enumerated type (EnumSet[Partition]), which can have one of the following values:

readgroup

sample

library

platform

center

sample_by_platform

sample_by_center

sample_by_platform_by_center

EnumSet[Partition]  [sample]

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--print-base-counts

Add base counts to per-locus output
Instead of reporting depth, the program will report the base pileup at each locus

boolean  false

---

--QUIET

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

Boolean  false

---

--read-filter / -RF

Read filters to be applied before analysis

List[String]  []

---

--read-index / -read-index

Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.

List[GATKPath]  []

---

--read-validation-stringency / -VS

Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value 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 --read-validation-stringency argument is an enumerated type (ValidationStringency), which can have one of the following values:

STRICT

LENIENT

SILENT

ValidationStringency  SILENT

---

--reference / -R

Reference sequence file

R GATKPath  null

---

--seconds-between-progress-updates / -seconds-between-progress-updates

Output traversal statistics every time this many seconds elapse

double  10.0  [ [ -∞  ∞ ] ]

---

--sequence-dictionary / -sequence-dictionary

Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.

GATKPath  null

---

--showHidden / -showHidden

display hidden arguments

boolean  false

---

--sites-only-vcf-output

If true, don't emit genotype fields when writing vcf file output.

boolean  false

---

--start

Starting (left endpoint) for granular binning
Sets the low-coverage cutoff for granular binning. All loci with depth < START are counted in the first bin.

int  1  [ [ 0  ∞ ] ]

---

--stop

Ending (right endpoint) for granular binning
Sets the high-coverage cutoff for granular binning. All loci with depth > STOP are counted in the last bin.

int  500  [ [ 1  ∞ ] ]

---

--summary-coverage-threshold

Coverage threshold (in percent) for summarizing statistics
For summary file outputs, report the percentage of bases covered to an amount equal to or greater than this number (e.g. % bases >= CT for each sample). Defaults to 15; can take multiple arguments.

List[Integer]  [15]

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--tmp-dir

Temp directory to use.

GATKPath  null

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--use-jdk-deflater / -jdk-deflater

Whether to use the JdkDeflater (as opposed to IntelDeflater)

boolean  false

---

--use-jdk-inflater / -jdk-inflater

Whether to use the JdkInflater (as opposed to IntelInflater)

boolean  false

---

--verbosity / -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 4.2.5.0-SNAPSHOT built at Mon, 7 Feb 2022 11:18:01 -0500.