Apply base quality score recalibration

Category Read Data Manipulation

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Overview

Apply base quality score recalibration

This tool performs the second pass in a two-stage process called Base Quality Score Recalibration (BQSR). Specifically, it recalibrates the base qualities of the input reads based on the recalibration table produced by the BaseRecalibrator tool, and outputs a recalibrated BAM or CRAM file.

Summary of the BQSR procedure

The goal of this procedure is to correct for systematic bias that affect the assignment of base quality scores by the sequencer. The first pass consists of calculating error empirically and finding patterns in how error varies with basecall features over all bases. The relevant observations are written to a recalibration table. The second pass consists of applying numerical corrections to each individual basecall based on the patterns identified in the first step (recorded in the recalibration table) and write out the recalibrated data to a new BAM or CRAM file.

Input

• A BAM or CRAM file containing input read data
• The covariates table (= recalibration file) generated by BaseRecalibrator on the input BAM or CRAM file

Output

A BAM or CRAM file containing the recalibrated read data

Usage example

 gatk ApplyBQSR \
   -R reference.fasta \
   -I input.bam \
   --bqsr-recal-file recalibration.table \
   -O output.bam
 

Notes

• This tool replaces the use of PrintReads for the application of base quality score recalibration as practiced in earlier versions of GATK (2.x and 3.x).
• You should only run ApplyBQSR with the covariates table created from the input BAM or CRAM file(s).
• Original qualities can be retained in the output file under the "OQ" tag if desired. See the `--emit-original-quals` argument for details.

ApplyBQSR 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
--bqsr-recal-file  -bqsr	null	Input recalibration table for BQSR
--input  -I	[]	BAM/SAM/CRAM file containing reads
--output  -O	null	Write output to this file
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.
--emit-original-quals	false	Emit original base qualities under the OQ tag
--gcs-max-retries  -gcs-retries	20	If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
--global-qscore-prior	-1.0	Global Qscore Bayesian prior to use for BQSR
--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
--preserve-qscores-less-than	6	Don't recalibrate bases with quality scores less than this threshold
--quantize-quals	0	Quantize quality scores to a given number of levels
--reference  -R	null	Reference sequence
--use-original-qualities  -OQ	false	Use the base quality scores from the OQ tag
--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-sequence-dictionary-validation	false	If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
--disable-tool-default-read-filters	false	Disable all tool default read filters
--exclude-intervals  -XL	[]	One or more genomic intervals to exclude from processing
--gatk-config-file	null	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
--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
--round-down-quantized	false	Round quals down to nearest quantized qual
--showHidden	false	display hidden arguments
--static-quantized-quals	[]	Use static quantized quality scores to a given number of levels (with -bqsr)

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 / NA

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

List[File]  []

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--bqsr-recal-file / -bqsr

Input recalibration table for BQSR
This argument is required for recalibration of base qualities. The recalibration table is a file produced by the BaseRecalibrator tool. Please be aware that you should only run recalibration with the recalibration file created on the same input data.

R File  null

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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  [ [ -∞  ∞ ] ]

---

--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

---

--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]  []

---

--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

boolean  false

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--emit-original-quals / NA

Emit original base qualities under the OQ tag
The tool is capable of writing out the original quality scores of each read in the recalibrated output file under the "OQ" tag. By default, this behavior is disabled because emitting original qualities results in a significant increase of the file size. Use this flag to turn on emission of original qualities.

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 / NA

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  [ [ -∞  ∞ ] ]

---

--global-qscore-prior / NA

Global Qscore Bayesian prior to use for BQSR
If specified, the value of this argument will be used as a flat prior for all mismatching quality scores instead of the reported quality score (assigned by the sequencer).

double  -1.0  [ [ -∞  ∞ ] ]

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--help / -h

display the help message

boolean  false

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--input / -I

BAM/SAM/CRAM file containing reads

R List[String]  []

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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  [ [ -∞  ∞ ] ]

---

--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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--output / -O

Write output to this file

R File  null

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--preserve-qscores-less-than / NA

Don't recalibrate bases with quality scores less than this threshold
This flag tells GATK not to modify quality scores less than this value. Instead they will be written out unmodified in the recalibrated BAM file. In general it's unsafe to change qualities scores below < 6, since base callers use these values to indicate random or bad bases. For example, Illumina writes Q2 bases when the machine has really gone wrong. This would be fine in and of itself, but when you select a subset of these reads based on their ability to align to the reference and their dinucleotide effect, your Q2 bin can be elevated to Q8 or Q10, leading to issues downstream.

int  6  [ [ -∞  ∞ ] ]

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--quantize-quals / NA

Quantize quality scores to a given number of levels
Turns on the base quantization module. It requires a recalibration report. A value of 0 here means "do not quantize". Any value greater than zero will be used to recalculate the quantization using that many levels. Negative values mean that we should quantize using the recalibration report's quantization level.

Exclusion: This argument cannot be used at the same time as static-quantized-quals, round-down-quantized.

int  0  [ [ -∞  ∞ ] ]

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--QUIET / NA

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]  []

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--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]  []

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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

String  null

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--round-down-quantized / NA

Round quals down to nearest quantized qual
Round down quantized only works with the static_quantized_quals option, and should not be used with the dynamic binning option provided by quantize_quals. When roundDown = false, rounding is done in probability space to the nearest bin. When roundDown = true, the value is rounded to the nearest bin that is smaller than the current bin.

Exclusion: This argument cannot be used at the same time as quantize-quals.

boolean  false

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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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--static-quantized-quals / NA

Use static quantized quality scores to a given number of levels (with -bqsr)
Static quantized quals are entirely separate from the quantize_qual option which uses dynamic binning. The two types of binning should not be used together.

Exclusion: This argument cannot be used at the same time as quantize-quals.

List[Integer]  []

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--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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--use-original-qualities / -OQ

Use the base quality scores from the OQ tag
This flag tells GATK to use the original base qualities (that were in the data before BQSR/recalibration) which are stored in the OQ tag, if they are present, rather than use the post-recalibration quality scores. If no OQ tag is present for a read, the standard quality score will be used.

Boolean  false

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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 4.0.1.2 built at 02-02-2019 02:02:55.