GetPileupSummaries

Tabulates pileup metrics for inferring contamination

Category Coverage Analysis

Overview

Summarizes counts of reads that support reference, alternate and other alleles for given sites. Results can be used with {@link CalculateContamination}.

The tool requires a common germline variant sites VCF, e.g. derived from the gnomAD resource, with population allele frequencies (AF) in the INFO field. This resource must contain only biallelic SNPs and can be an eight-column sites-only VCF. The tool ignores the filter status of the variant calls in this germline resource.

This tool is featured in the Somatic Short Mutation calling Best Practice Workflow. See Tutorial#11136 for a step-by-step description of the workflow and Article#11127 for an overview of what traditional somatic calling entails. For the latest pipeline scripts, see the Mutect2 WDL scripts directory. In particular, the mutect_resources.wdl script prepares a suitable resource from a larger dataset. An example excerpt is shown.

 #CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO
 chr6	29942512	.	G	C	2974860	VQSRTrancheSNP99.80to99.90	AF=0.063
 chr6	29942517	.	C	A	2975860	VQSRTrancheSNP99.80to99.90	AF=0.062
 chr6	29942525	.	G	C	2975600	VQSRTrancheSNP99.60to99.80	AF=0.063
 chr6	29942547	rs114945359	G	C	15667700	PASS	AF=0.077

Usage examples

 gatk GetPileupSummaries \
   -I tumor.bam \
   -V common_biallelic.vcf.gz \
   -L common_biallelic.vcf.gz \
   -O pileups.table

 gatk GetPileupSummaries \
   -I normal.bam \
   -V common_biallelic.vcf.gz \
   -L common_biallelic.vcf.gz \
   -O pileups.table

Although the sites (-L) and variants (-V) resources will often be identical, this need not be the case. For example,

 gatk GetPileupSummaries \
   -I normal.bam \
   -V gnomad.vcf.gz \
   -L common_snps.interval_list \
   -O pileups.table

attempts to get pileups at a list of common snps and emits output for those sites that are present in gnomAD, using the allele frequencies from gnomAD. Note that the sites may be a subset of the variants, the variants may be a subset of the sites, or they may overlap partially. In all cases pileup summaries are emitted for the overlap and nowhere else. The most common use case in which sites and variants differ is when the variants resources is a large file and the sites is an interval list subset from that file.

GetPileupSummaries tabulates results into six columns as shown below. The alt_count and allele_frequency correspond to the ALT allele in the germline resource.

 contig	position	ref_count	alt_count	other_alt_count	allele_frequency
 chr6	29942512	9	0	0	0.063
 chr6	29942517	13	1	0	0.062
 chr6	29942525	13	7	0	0.063
 chr6	29942547	36	0	0	0.077

Note the default maximum population AF ({@code --maximum-population-allele-frequency} or {@code -max-af}) is set to 0.2, which limits the sites the tool considers to those in the variants resource file that have AF of 0.2 or less. Likewise, the default minimum population AF ({@code --minimum-population-allele-frequency} or {@code -min-af}) is set to 0.01, which limits the sites the tool considers to those in the variants resource file that have AF of 0.01 or more.

Finally for those using mappers other than bwa mem or dragen-os, {@code --minimum-mapping-quality} threshold is set to 50, which limits the usable reads that tool considers for generating pileups. Certain mappers are known to assign scores less that this threshold even for the unique mappings. If you observe all empty results in your summary file please adjust the {@code --minimum-mapping-quality} parameter according to your input files.

Additional Information

Read filters

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

GetPileupSummaries 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		The output table
--variant -V		A VCF file containing variants and allele frequencies
Optional Tool Arguments
--arguments_file		read one or more arguments files and add them to the command line
--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).
--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-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.
--maximum-population-allele-frequency -max-af	0.2	Maximum population allele frequency of sites to consider.
--minimum-population-allele-frequency -min-af	0.01	Minimum population allele frequency of sites to consider. A low value increases accuracy at the expense of speed.
--reference -R		Reference sequence
--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
--inverted-read-filter -XRF		Inverted (with flipped acceptance/failure conditions) read filters applied before analysis (after regular read filters).
--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
--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.

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

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

boolean true

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

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

boolean true

--arguments_file

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

List[File] []

--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 [ [ -∞ ∞ ] ]

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

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

boolean true

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

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

boolean false

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

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

boolean true

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

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

boolean false

--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

--disable-read-filter / -DF

Read filters to be disabled before analysis

List[String] []

--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

--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

--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). strings gathered from the command line -XL argument to be parsed into intervals to exclude

List[String] []

--gatk-config-file

A configuration file to use with the GATK.

String null

--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 [ [ -∞ ∞ ] ]

--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 ""

--help / -h

display the help message

boolean false

--input / -I

BAM/SAM/CRAM file containing reads

R List[GATKPath] []

--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 [ [ -∞ ∞ ] ]

--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

--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 [ [ -∞ ∞ ] ]

--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

--intervals / -L

One or more genomic intervals over which to operate

R List[String] []

--inverted-read-filter / -XRF

Inverted (with flipped acceptance/failure conditions) read filters applied before analysis (after regular read filters).

List[String] []

--lenient / -LE

Lenient processing of VCF files

boolean false

--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 ∞ ] ]

--maximum-population-allele-frequency / -max-af

Maximum population allele frequency of sites to consider.

double 0.2 [ [ -∞ ∞ ] ]

--minimum-population-allele-frequency / -min-af

Minimum population allele frequency of sites to consider. A low value increases accuracy at the expense of speed.

double 0.01 [ [ -∞ ∞ ] ]

--output / -O

The output table

R File null

--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: