Apply base quality score recalibration
Category Read Data Manipulation
Overview
Apply base quality score recalibrationThis 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.
Additional Information
Read filters
This Read Filter is automatically applied to the data by the Engine before processing by ApplyBQSR.
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 |
Input recalibration table for BQSR | ||
--input -I |
BAM/SAM/CRAM file containing reads | ||
--output -O |
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. | |
--disable-sequence-dictionary-validation |
false | If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk! | |
--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 | |
--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. | ||
--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 |
Reference sequence | ||
--sites-only-vcf-output |
false | If true, don't emit genotype fields when writing vcf file output. | |
--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-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 | |||
--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.
--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] []
--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
--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
--emit-original-quals
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
--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 ""
--global-qscore-prior
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 [ [ -∞ ∞ ] ]
--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
List[String] []
--lenient / -LE
Lenient processing of VCF files
boolean false
--max-variants-per-shard
If non-zero, partitions VCF output into shards, each containing up to the given number of records.
int 0 [ [ 0 ∞ ] ]
--output / -O
Write output to this file
R GATKPath null
--preserve-qscores-less-than
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 [ [ 0 [ 6 ∞ ] ]
--quantize-quals
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 [ [ -∞ ∞ ] ]
--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
GATKPath null
--round-down-quantized
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
--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
--static-quantized-quals
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] []
--tmp-dir
Temp directory to use.
GATKPath null
--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
--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
--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
--version
display the version number for this tool
boolean false
GATK version 4.4.0.0 built at Thu, 16 Mar 2023 15:00:37 -0400.
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