Apply a score cutoff to filter variants based on a recalibration table
This tool performs the second pass in a two-stage process called Variant Quality Score Recalibration (VQSR). Specifically, it applies filtering to the input variants based on the recalibration table produced in the first step by VariantRecalibrator and a target sensitivity value, which the tool matches internally to a VQSLOD score cutoff based on the model's estimated sensitivity to a set of true variants.
The filter determination is not just a pass/fail process. The tool evaluates for each variant which "tranche", or slice of the dataset, it falls into in terms of sensitivity to the truthset. Variants in tranches that fall below the specified truth sensitivity filter level have their FILTER field annotated with the corresponding tranche level. This results in a callset that is filtered to the desired level but retains the information necessary to increase sensitivity if needed.
To be clear, please note that by "filtered", we mean that variants failing the requested tranche cutoff are marked as filtered in the output VCF; they are not discarded unless the option to do so is specified.
The purpose of variant recalibration is to assign a well-calibrated probability to each variant call in a call set. These probabilities can then be used to filter the variants with a greater level of accuracy and flexibility than can typically be achieved by traditional hard-filter (filtering on individual annotation value thresholds). The first pass consists of building a model that describes how variant annotation values co-vary with the truthfulness of variant calls in a training set, and then scoring all input variants according to the model. The second pass simply consists of specifying a target sensitivity value (which corresponds to an empirical VQSLOD cutoff) and applying filters to each variant call according to their ranking. The result is a VCF file in which variants have been assigned a score and filter status.
VQSR is probably the hardest part of the Best Practices to get right, so be sure to read the method documentation, parameter recommendations and tutorial to really understand what these tools do and how to use them for best results on your own data.
gatk ApplyVQSR \ -R Homo_sapiens_assembly38.fasta \ -V input.vcf.gz \ -O output.vcf.gz \ --truth-sensitivity-filter-level 99.0 \ --tranches-file output.tranches \ --recal-file output.recal \ -mode SNP
gatk ApplyVQSR \ -R Homo_sapiens_assembly38.fasta \ -V input.vcf.gz \ -O output.vcf.gz \ -AS \ --truth-sensitivity-filter-level 99.0 \ --tranches-file output.AS.tranches \ --recal-file output.AS.recal \ -mode SNP
Note that the tranches and recalibration files must have been produced by an allele-specific run of VariantRecalibrator. Also note that the AS_culprit, AS_FilterStatus, and AS_VQSLOD fields will have placeholder values (NA or NaN) for alleles of a type that have not yet been processed by ApplyVQSR. The spanning deletion allele (*) will not be recalibrated because it represents missing data. Its VQSLOD will remain NaN, and its culprit and FilterStatus will be NA.
Each allele will be annotated by its corresponding entry in the AS_FilterStatus INFO field annotation. Allele-specific VQSLOD and culprit are also carried through from VariantRecalibrator, and stored in the AS_VQSLOD and AS_culprit INFO fields, respectively. The site-level filter is set to the most lenient of any of the allele filters. That is, if one allele passes, the whole site will be PASS. If no alleles pass, the site-level filter will be set to the lowest sensitivity tranche among all the alleles.
This Read Filter is automatically applied to the data by the Engine before processing by ApplyVQSR.
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 |
The output filtered and recalibrated VCF file in which each variant is annotated with its VQSLOD value | ||
| --recal-file |
The input recal file used by ApplyVQSR | ||
| --variant -V |
One or more VCF files containing variants | ||
| 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! | |
| --exclude-filtered |
false | Don't output filtered loci after applying the recalibration | |
| --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 | |
| --ignore-all-filters |
false | If specified, the variant recalibrator will ignore all input filters. Useful to rerun the VQSR from a filtered output file. | |
| --ignore-filter |
If specified, the recalibration will be applied to variants marked as filtered by the specified filter name in the input VCF file | ||
| --interval-merging-rule -imr |
ALL | Interval merging rule for abutting intervals | |
| --intervals -L |
One or more genomic intervals over which to operate | ||
| --mode |
SNP | Recalibration mode to employ: 1.) SNP for recalibrating only SNPs (emitting indels untouched in the output VCF); 2.) INDEL for indels; and 3.) BOTH for recalibrating both SNPs and indels simultaneously. | |
| --reference -R |
Reference sequence | ||
| --sites-only-vcf-output |
false | If true, don't emit genotype fields when writing vcf file output. | |
| --tranches-file |
The input tranches file describing where to cut the data | ||
| --truth-sensitivity-filter-level -ts-filter-level |
The truth sensitivity level at which to start filtering | ||
| --use-allele-specific-annotations -AS |
false | If specified, the tool will attempt to apply a filter to each allele based on the input tranches and allele-specific .recal file. | |
| --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 | |
| --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 | |||
| --lod-score-cutoff |
The VQSLOD score below which to start filtering | ||
| --showHidden |
false | display hidden arguments | |
| --variant-output-filtering |
Restrict the output variants to ones that match the specified intervals according to the specified matching mode. | ||
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.
If true, adds a PG tag to created SAM/BAM/CRAM files.
boolean true
If true, adds a command line header line to created VCF files.
boolean true
read one or more arguments files and add them to the command line
List[File] []
Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.
int -1 [ [ -∞ ∞ ] ]
Size of the cloud-only prefetch buffer (in MB; 0 to disable).
int 40 [ [ -∞ ∞ ] ]
If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.
boolean true
If true, create a MD5 digest for any BAM/SAM/CRAM file created
boolean false
If true, create a VCF index when writing a coordinate-sorted VCF file.
boolean true
If true, create a a MD5 digest any VCF file created.
boolean 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.
boolean false
Read filters to be disabled before analysis
List[String] []
If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
boolean false
Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)
boolean false
Don't output filtered loci after applying the recalibration
boolean false
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] []
A configuration file to use with the GATK.
String null
If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
int 20 [ [ -∞ ∞ ] ]
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 ""
display the help message
boolean false
If specified, the variant recalibrator will ignore all input filters. Useful to rerun the VQSR from a filtered output file.
boolean false
If specified, the recalibration will be applied to variants marked as filtered by the specified filter name in the input VCF file
For this to work properly, the --ignore-filter argument should also be applied to the VariantRecalibration command.
List[String] []
BAM/SAM/CRAM file containing reads
List[GATKPath] []
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 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:
IntervalMergingRule ALL
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 [ [ -∞ ∞ ] ]
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:
IntervalSetRule UNION
One or more genomic intervals over which to operate
List[String] []
Inverted (with flipped acceptance/failure conditions) read filters applied before analysis (after regular read filters).
List[String] []
Lenient processing of VCF files
boolean false
The VQSLOD score below which to start filtering
Double null
If non-zero, partitions VCF output into shards, each containing up to the given number of records.
int 0 [ [ 0 ∞ ] ]
Recalibration mode to employ: 1.) SNP for recalibrating only SNPs (emitting indels untouched in the output VCF); 2.) INDEL for indels; and 3.) BOTH for recalibrating both SNPs and indels simultaneously.
The --mode argument is an enumerated type (Mode), which can have one of the following values:
Mode SNP
The output filtered and recalibrated VCF file in which each variant is annotated with its VQSLOD value
R GATKPath null
Whether to suppress job-summary info on System.err.
Boolean false
Read filters to be applied before analysis
List[String] []
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] []
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:
ValidationStringency SILENT
The input recal file used by ApplyVQSR
R FeatureInput[VariantContext] null
Reference sequence
GATKPath null
Output traversal statistics every time this many seconds elapse
double 10.0 [ [ -∞ ∞ ] ]
Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
GATKPath null
display hidden arguments
boolean false
If true, don't emit genotype fields when writing vcf file output.
boolean false
Temp directory to use.
GATKPath null
The input tranches file describing where to cut the data
GATKPath null
The truth sensitivity level at which to start filtering
Double null
If specified, the tool will attempt to apply a filter to each allele based on the input tranches and allele-specific .recal file.
Filter the input file based on allele-specific recalibration data. See tool docs for site-level and allele-level filtering details.
Requires a .recal file produced using an allele-specific run of VariantRecalibrator.
boolean false
Whether to use the JdkDeflater (as opposed to IntelDeflater)
boolean false
Whether to use the JdkInflater (as opposed to IntelInflater)
boolean false
One or more VCF files containing variants
R List[GATKPath] []
Restrict the output variants to ones that match the specified intervals according to the specified matching mode.
The --variant-output-filtering argument is an enumerated type (Mode), which can have one of the following values:
Mode null
Control verbosity of logging.
The --verbosity argument is an enumerated type (LogLevel), which can have one of the following values:
LogLevel INFO
display the version number for this tool
boolean false
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GATK version 4.6.2.0 built at Sun, 13 Apr 2025 13:21:43 -0400.