Make a panel of normals for use with Mutect2

Category Variant Filtering

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Overview

Create a panel of normals (PoN) containing germline and artifactual sites for use with Mutect2.

The tool takes multiple normal sample callsets produced by {@link Mutect2}'s tumor-only mode and collates sites present in multiple samples (two by default, set by the --min-sample-count argument) into a sites-only VCF. The PoN captures common artifacts. Mutect2 then uses the PoN to filter variants at the site-level. The --max-germline-probability argument sets the threshold for possible germline variants to be included in the PoN. By default this is set to 0.5, so that likely germline events are excluded. This is usually the correct behavior as germline variants are best handled by probabilistic modeling via Mutect2's --germline-resource argument. A germline resource, such as gnomAD in the case of humans, is a much more refined tool for germline filtering than any PoN could be.

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.

Example workflow

Step 1. Run Mutect2 in tumor-only mode for each normal sample.

Note that as of May, 2019 -max-mnp-distance must be set to zero to avoid a bug in GenomicsDBImport.

 gatk Mutect2 -R reference.fasta -I normal1.bam -max-mnp-distance 0 -O normal1.vcf.gz
 

Step 2. Create a GenomicsDB from the normal Mutect2 calls.

    gatk GenomicsDBImport -R reference.fasta -L intervals.interval_list \
       --genomicsdb-workspace-path pon_db \
       -V normal1.vcf.gz \
       -V normal2.vcf.gz \
       -V normal3.vcf.gz
  

Step 3. Combine the normal calls using CreateSomaticPanelOfNormals.

 gatk CreateSomaticPanelOfNormals -R reference.fasta -V gendb://pon_db -O pon.vcf.gz
 

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Additional Information

Read filters

This Read Filter is automatically applied to the data by the Engine before processing by CreateSomaticPanelOfNormals.

• WellformedReadFilter

CreateSomaticPanelOfNormals 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
--output  -O		Output vcf
--variant  -V		A VCF file containing variants
Optional Tool Arguments
--arguments_file		read one or more arguments files and add them to the command line
--call-genotypes	false	Output called genotypes in final VCF (otherwise no-call)
--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.
--genomicsdb-max-alternate-alleles	50	Maximum number of alternate alleles that will be combined on reading from GenomicsDB
--genomicsdb-shared-posixfs-optimizations	false	Allow for optimizations to improve the usability and performance for shared Posix Filesystems(e.g. NFS, Lustre). If set, file level locking is disabled and file system writes are minimized.
--genomicsdb-use-gcs-hdfs-connector	false	Use the GCS HDFS Connector instead of the native GCS SDK client with gs:// URLs.
--germline-resource		Population vcf of germline sequencing containing allele fractions.
--help  -h	false	display the help message
--interval-merging-rule  -imr	ALL	Interval merging rule for abutting intervals
--intervals  -L		One or more genomic intervals over which to operate
--max-germline-probability	0.5	Skip genotypes with germline probability greater than this value
--min-sample-count	2	Number of samples containing a variant site required to include it in the panel of normals.
--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.
--input  -I		BAM/SAM/CRAM file containing reads
--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
--genomicsdb-use-bcf-codec	false	Use BCF Codec Streaming for data from GenomicsDB instead of the default VCFCodec. BCFCodec performs slightly better but currently does not support 64-bit width positions and INFO fields and for computed annotation sizes to exceed 32-bit integer space.
--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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--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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--call-genotypes

Output called genotypes in final VCF (otherwise no-call)
Output called genotypes in the final VCF (otherwise no-call)

boolean  false

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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  [ [ -∞  ∞ ] ]

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--cloud-prefetch-buffer / -CPB

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

int  40  [ [ -∞  ∞ ] ]

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

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  [ [ -∞  ∞ ] ]

---

--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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--genomicsdb-max-alternate-alleles

Maximum number of alternate alleles that will be combined on reading from GenomicsDB
Maximum number of alternate alleles that will report likelihoods after being combined on reading from GenomicsDB (including ) Must be at least one greater than the maximum number of alternate alleles for genotyping. A typical value is 3 more than the --max-alternate-alleles value that's used by GenotypeGVCFs and larger differences result in more robustness to PCR-related indel errors. NOTE: GenotypeGVCFs will drop multi-allelic sites with more than this many alternate alleles since they are missing likelihoods. See also

int  50  [ [ -∞  ∞ ] ]

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--genomicsdb-shared-posixfs-optimizations

Allow for optimizations to improve the usability and performance for shared Posix Filesystems(e.g. NFS, Lustre). If set, file level locking is disabled and file system writes are minimized.

boolean  false

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--genomicsdb-use-bcf-codec

Use BCF Codec Streaming for data from GenomicsDB instead of the default VCFCodec. BCFCodec performs slightly better but currently does not support 64-bit width positions and INFO fields and for computed annotation sizes to exceed 32-bit integer space.
Currently there is no support for 64-bit fields in BCF2Codec. The VCFCodec allows for 64-bit width positions and INFO fields and for computed annotation sizes to exceed the 32-bit integer space while encoding/decoding with GenomicsDB. Use the BCF2Codec option if and only if performance is an issue.

boolean  false

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--genomicsdb-use-gcs-hdfs-connector

Use the GCS HDFS Connector instead of the native GCS SDK client with gs:// URLs.

boolean  false

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--germline-resource

Population vcf of germline sequencing containing allele fractions.
A resource, such as gnomAD, containing population allele frequencies of common and rare variants. We use this to remove germline variants from the panel of normals, keeping only technical artifacts

FeatureInput[VariantContext]  null

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--help / -h

display the help message

boolean  false

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--input / -I

BAM/SAM/CRAM file containing reads

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

List[String]  []

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--lenient / -LE

Lenient processing of VCF files

boolean  false

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--max-germline-probability

Skip genotypes with germline probability greater than this value

double  0.5  [ [ -∞  ∞ ] ]

---

--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-sample-count

Number of samples containing a variant site required to include it in the panel of normals.

int  2  [ [ -∞  ∞ ] ]

---

--output / -O

Output vcf

R String  null

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--QUIET

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

Boolean  false

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--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]  []

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--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

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--reference / -R

Reference sequence

GATKPath  null

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

Output traversal statistics every time this many seconds elapse

double  10.0  [ [ -∞  ∞ ] ]

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--sequence-dictionary / -sequence-dictionary

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

GATKPath  null

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--showHidden / -showHidden

display hidden arguments

boolean  false

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--sites-only-vcf-output

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

boolean  false

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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

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

Whether to use the JdkInflater (as opposed to IntelInflater)

boolean  false

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--variant / -V

A VCF file containing variants

R GATKPath  null

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--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 4.4.0.0 built at Thu, 16 Mar 2023 15:00:37 -0400.