Filter Mutect2 somatic variant calls using orientation bias

Category Variant Filtering

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Overview

Additionally filter Mutect2 somatic variant calls for sequence context-dependent artifacts, e.g. OxoG or FFPE deamination.

This tool is complementary to FilterMutectCalls and if run, should be run after FilterMutectCalls. The tool requires the pre-adapter detailed metrics calculated by Picard CollectSequencingArtifactMetrics and specification of the base substitution(s) to consider for orientation bias. For a given base substitution specified with the --artifact-modes argument, the tool considers both the forward and reverse complement for filtering. That is, specifying --artifact-modes 'G/T' means the tool considers G to T and A to C artifacts for filtering.

The metrics from CollectSequencingArtifactMetrics give a global view across a sample's genome that empowers decision making in ways site-specific analyses cannot. The detailed metrics measure orientation bias for all three-base contexts and help determine whether variant filtering for a sequence context is necessary.

The most common artifact to consider for filtering is the OxoG (8-Oxoguanine) artifact. OxoG artifacts stem from oxidation of guanine to 8-oxoguanine, which results in G to T transversions. If samples were derived from histological slides, then consider the FFPE (formalin-fixed paraffin-embedded) artifact. FFPE artifacts stem from formaldehyde deamination of cytosines, which results in C to T transitions.

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 CollectSequencingArtifactMetrics on sample BAM.

Either invoke CollectSequencingArtifactMetrics using the GATK4 launch script:

 gatk CollectSequencingArtifactMetrics \
   -R reference.fa \
   -I tumor.bam \
   --FILE_EXTENSION ".txt" \
   -O tumor_artifact
 

Or run CollectSequencingArtifactMetrics from a standalone Picard jar:

 java -jar picard.jar CollectSequencingArtifactMetrics \
   R=reference.fa \
   I=tumor.bam \
   --FILE_EXTENSION=.txt \
   O=tumor_artifact

This generates a number of metrics files, including tumor_artifact.pre_adapter_detail_metrics.txt.

Step 2. Run FilterByOrientationBias on Mutect2 calls that have been filtered by FilterMutectCalls.

Here we specify the G to T OxoG artifact and provide the pre_adapter_detail_metrics from step 1.

 gatk FilterByOrientationBias \
   -V filtered.vcf.gz \
   --artifact-modes 'G/T' \
   -P tumor_artifact.pre_adapter_detail_metrics.txt \
   -O oxog_filtered.vcf.gz
 

This produces variant calls filtered with the orientation_bias filter and a summary oxog_filtered.vcf.gz.summary file. The summary tallies the number of calls for the sequence context(s) and the number of those that the tool filtered.

Further points of interest

• The tool only considers passing calls and relies on the presence of sample-level F1R2 and F2R1 annotations.
• If a site is multiallelic, only the first alternate allele is considered in filtering. If a sample call is triallelic, it may not be considered for filtering. The implication is that FilterByOrientationBias is only recommended for use with Mutect2 callsets from originally diploid and non-pooled samples.
• For calls that the tool filters, the FILTER and sample fields are each annotated with the 'orientation_bias' label.

FilterByOrientationBias 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	null	Output Somatic SNP/Indel VCF file
--pre-adapter-detail-file  -P	null	PreAdapter Detail metrics file. Usually, generated by CollectSequencingArtifactMetrics.
--variant  -V	null	A VCF file containing variants
Optional Tool Arguments
--arguments_file	[]	read one or more arguments files and add them to the command line
--artifact-modes  -AM	[]	PreAdapter Detail artifacts of interest on the forward strand. 'C/A' for a single artifact. Specify this parameter multiple times to process multiple artifacts at the same time: 'C/A,T/G' Artifacts must be one base to one base (e.g. 'CC/CA' is illegal). G>T is OxoG.
--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
--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
--reference  -R	null	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	null	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
--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	null	Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--TMP_DIR	[]	Undocumented option
--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 / NA

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

List[File]  []

---

--artifact-modes / -AM

PreAdapter Detail artifacts of interest on the forward strand. 'C/A' for a single artifact. Specify this parameter multiple times to process multiple artifacts at the same time: 'C/A,T/G' Artifacts must be one base to one base (e.g. 'CC/CA' is illegal). G>T is OxoG.

List[String]  []

---

--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

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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]  []

---

--gatk-config-file / NA

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  [ [ -∞  ∞ ] ]

---

--help / -h

display the help message

boolean  false

---

--input / -I

BAM/SAM/CRAM file containing reads

List[String]  []

---

--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

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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  [ [ -∞  ∞ ] ]

---

--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]  []

---

--lenient / -LE

Lenient processing of VCF files

boolean  false

---

--output / -O

Output Somatic SNP/Indel VCF file

R File  null

---

--pre-adapter-detail-file / -P

PreAdapter Detail metrics file. Usually, generated by CollectSequencingArtifactMetrics.

R File  null

---

--QUIET / NA

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[String]  []

---

--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

String  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  [ [ -∞  ∞ ] ]

---

--sequence-dictionary / -sequence-dictionary

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

String  null

---

--showHidden / -showHidden

display hidden arguments

boolean  false

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--sites-only-vcf-output / NA

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

boolean  false

---

--TMP_DIR / NA

Undocumented option

List[File]  []

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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 String  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 / 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.