CallableLoci (EXPERIMENTAL) – GATK

Determine callable status of loci

Category Coverage Analysis

Overview

Collect statistics on callable, uncallable, poorly mapped, and other parts of the genome

A very common question about a NGS set of reads is what areas of the genome are considered callable. This tool considers the coverage at each locus and emits either a per base state or a summary interval BED file that partitions the genomic intervals into the following callable states:

REF_N: The reference base was an N, which is not considered callable the GATK
PASS: The base satisfied the min. depth for calling but had less than maxDepth to avoid having EXCESSIVE_COVERAGE
NO_COVERAGE: Absolutely no reads were seen at this locus, regardless of the filtering parameters
LOW_COVERAGE: There were fewer than min. depth bases at the locus, after applying filters
EXCESSIVE_COVERAGE: More than --max-depth read at the locus, indicating some sort of mapping problem
POOR_MAPPING_QUALITY: More than --max-fraction-of-reads-with-low-mapq at the locus, indicating a poor mapping quality of the reads

Input

A BAM file containing exactly one sample.

Output

A file with the callable status covering each base and a table of callable status x count of all examined bases

Usage example

  gatk CallableLoci \
      -I myreads.bam \
      -R myreference.fasta \
      -O callable_status.bed \    
      --summary table.txt

would produce a BED file that looks like:

     20 10000000 10000864 PASS
     20 10000865 10000985 POOR_MAPPING_QUALITY
     20 10000986 10001138 PASS
     20 10001139 10001254 POOR_MAPPING_QUALITY
     20 10001255 10012255 PASS
     20 10012256 10012259 POOR_MAPPING_QUALITY
     20 10012260 10012263 PASS
     20 10012264 10012328 POOR_MAPPING_QUALITY
     20 10012329 10012550 PASS
     20 10012551 10012551 LOW_COVERAGE
     20 10012552 10012554 PASS
     20 10012555 10012557 LOW_COVERAGE
     20 10012558 10012558 PASS

as well as a summary table that looks like:

                        state nBases
                        REF_N 0
                         PASS 996046
                  NO_COVERAGE 121
                 LOW_COVERAGE 928
           EXCESSIVE_COVERAGE 0
         POOR_MAPPING_QUALITY 2906

@author Mark DePristo / Jonn Smith @since May 7, 2010 / Nov 1, 2024

Additional Information

Read filters

These Read Filters are automatically applied to the data by the Engine before processing by CallableLoci.

CallableLoci 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
--input -I		BAM/SAM/CRAM file containing reads
--output -O		Output file (BED or per-base format)
--reference -R		Reference sequence file
--summary		Name of file for output summary
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!
--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
--interval-merging-rule -imr	ALL	Interval merging rule for abutting intervals
--intervals -L		One or more genomic intervals over which to operate
--max-depth		Maximum read depth before a locus is considered poorly mapped
--max-depth-per-sample	0	Maximum number of reads to retain per sample per locus. Reads above this threshold will be downsampled. Set to 0 to disable.
--max-fraction-of-reads-with-low-mapq -frlmq	0.1	If the fraction of reads at a base with low mapping quality exceeds this value, the site may be poorly mapped
--max-low-mapq -mlmq	1	Maximum value for MAPQ to be considered a problematic mapped read
--min-base-quality -mbq	20	Minimum quality of bases to count towards depth
--min-mapping-quality -mmq	10	Minimum mapping quality of reads to count towards depth
--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.
--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
--format	BED	Output format
--min-depth	4	Minimum QC+ read depth before a locus is considered callable
--min-depth-for-low-mapq -mdflmq	10	Minimum read depth before a locus is considered a potential candidate for poorly mapped
--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

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

List[File] []

--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

--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] []

--format / -format

Output format
The output of this tool will be written in this format. The recommended option is BED.

The --format argument is an enumerated type (OutputFormat), which can have one of the following values:

BED
STATE_PER_BASE

OutputFormat BED

--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 ""

--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] []

--inverted-read-filter / -XRF

Inverted (with flipped acceptance/failure conditions) read filters applied before analysis (after regular read filters).

List[String] []

--lenient / -LE

Lenient processing of VCF files

boolean false

--max-depth / -max-depth

Maximum read depth before a locus is considered poorly mapped
If the QC+ depth exceeds this value the site is considered to have EXCESSIVE_DEPTH

Integer null

--max-depth-per-sample / -max-depth-per-sample

Maximum number of reads to retain per sample per locus. Reads above this threshold will be downsampled. Set to 0 to disable.

int 0 [ [ -∞ ∞ ] ]

--max-fraction-of-reads-with-low-mapq / -frlmq

If the fraction of reads at a base with low mapping quality exceeds this value, the site may be poorly mapped
If the number of reads at this site is greater than minDepthForLowMAPQ and the fraction of reads with low mapping quality exceeds this fraction then the site has POOR_MAPPING_QUALITY.

double 0.1 [ [ -∞ ∞ ] ]

--max-low-mapq / -mlmq

Maximum value for MAPQ to be considered a problematic mapped read
The gap between this value and mmq are reads that are not sufficiently well mapped for calling but aren't indicative of mapping problems. For example, if maxLowMAPQ = 1 and mmq = 20, then reads with MAPQ == 0 are poorly mapped, MAPQ >= 20 are considered as contributing to calling, where reads with MAPQ >= 1 and 20 are not bad in and of themselves but aren't sufficiently good to contribute to calling. In effect this reads are invisible, driving the base to the NO_ or LOW_COVERAGE states

int 1 [ [ 0 255 ] ]

--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-base-quality / -mbq

Minimum quality of bases to count towards depth
Bases with less than minBaseQuality are viewed as not sufficiently high quality to contribute to the PASS state

int 20 [ [ 0 255 ] ]

--min-depth / -min-depth

Minimum QC+ read depth before a locus is considered callable
If the number of QC+ bases (on reads with MAPQ > minMappingQuality and with base quality > minBaseQuality) exceeds this value and is less than maxDepth the site is considered PASS.

int 4 [ [ 0 ∞ ] ]

--min-depth-for-low-mapq / -mdflmq

Minimum read depth before a locus is considered a potential candidate for poorly mapped
We don't want to consider a site as POOR_MAPPING_QUALITY just because it has two reads, and one is MAPQ. We won't assign a site to the POOR_MAPPING_QUALITY state unless there are at least minDepthForLowMAPQ reads covering the site.

int 10 [ [ -∞ ∞ ] ]

--min-mapping-quality / -mmq

Minimum mapping quality of reads to count towards depth
Reads with MAPQ > minMappingQuality are treated as usable for variation detection, contributing to the PASS state.

int 10 [ [ 0 255 ] ]

--output / -O

Output file (BED or per-base format)

R GATKPath null

--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 file

R GATKPath null

--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

--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

--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

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Articles in this section

Category Coverage Analysis

Overview

Input

Output

Usage example

Additional Information

Read filters

CallableLoci specific arguments

Argument details

--add-output-sam-program-record / -add-output-sam-program-record

--add-output-vcf-command-line / -add-output-vcf-command-line

--cloud-index-prefetch-buffer / -CIPB

--cloud-prefetch-buffer / -CPB

--create-output-bam-index / -OBI

--create-output-bam-md5 / -OBM

--create-output-variant-index / -OVI

--create-output-variant-md5 / -OVM

--disable-bam-index-caching / -DBIC

--disable-read-filter / -DF

--disable-sequence-dictionary-validation / -disable-sequence-dictionary-validation

--disable-tool-default-read-filters / -disable-tool-default-read-filters

--exclude-intervals / -XL

--format / -format

--gcs-max-retries / -gcs-retries

--help / -h

--input / -I

--interval-exclusion-padding / -ixp

--interval-merging-rule / -imr

--interval-padding / -ip

--interval-set-rule / -isr

--intervals / -L

--inverted-read-filter / -XRF

--lenient / -LE

--max-depth / -max-depth

--max-depth-per-sample / -max-depth-per-sample

--max-fraction-of-reads-with-low-mapq / -frlmq

--max-low-mapq / -mlmq

--min-base-quality / -mbq

--min-depth / -min-depth

--min-depth-for-low-mapq / -mdflmq

--min-mapping-quality / -mmq

--output / -O

--read-filter / -RF

--read-index / -read-index

--read-validation-stringency / -VS

--reference / -R

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

--sequence-dictionary / -sequence-dictionary

--showHidden / -showHidden

--use-jdk-deflater / -jdk-deflater

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

--verbosity / -verbosity

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