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ImmunoGenotyper

Provides genotyping summary for complex multigenic loci, like KIR or MHC.

Category Development/Pre-production Tools

Overview

This tool will generate genotype calls for complex loci (such as many immune genes), from next-generation sequence data. In general, most next-generation sequencing tasks (WGS, RNA-seq, etc.) expect to align each read uniquely in the genome and frequently discard or ignore those reads that do not. While this is sufficient for much of the genome, complex, polygenic regions are frequently systematically filtered or missed by many variant-calling or genotyping methods. Further, the reference genome often has a poor representation of complex loci, including those that are polygenic or have copy-number differences in the population (often the reference genome has a single copy of the gene), or situations where the set of genes varies widely between individuals (in which case a single reference genome poorly captures variation). This tool applies the same general approach as originally published for macaque MHC genotyping (https://www.ncbi.nlm.nih.gov/pubmed/19820716), though is a much newer implementation. This tool can either be run using targeted sequencing (i.e. amplicon), or using whole genome DNA- or RNA-seq data. To use this tool, first align your raw reads to a specialized database. For MHC, KIR, or NCR genotyping, we build a specialized database of alleles (IPD is a good starting point). We often use an aligner that will retain multiple matches per read (MOSAIK, for example); however, we use BWA-mem in some cases as well. This BAM is the input for the this tool. ImmunoGenotyper iterates this BAM, capturing the set of contigs each read aligned against, within the provided number of mismatches. We expect ambiguity in many cases, with reads aligning to groups of related alleles (i.e. A002*001:02 and A*002*001:01). Finally, we series of filters can be run (described for each argument below) that attempt to collapse these genotypes. A final table is produced. Note: the reference data is essential for this tool to operate as-expected, and thoughtful design of that database for your application is critical.

Usage example:

  java -jar DISCVRseq.jar ImmunoGenotyper \
     -R reference.fasta \
     -I myBam.bam \
     -mm 1 \
     -minReadCountForRef 10 \
     -O outputBaseDir

Additional Information

Genome/Reference Files

Please note that if this tools uses a reference genome, that FASTA must be indexed with samtools and to have a sequence dictionary created with Picard. See here for more information

Read filters

This Read Filter is automatically applied to the data by the Engine before processing by ImmunoGenotyper.

Filter

ImmunoGenotyper 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	null	Prefix for output files
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
--minAlignmentLength	40	Alignments shorted than this value will be discarded.
--minMappingQuality -mmq	0	Minimum mapping quality of reads to count towards depth.
--minPctForExport	0.0	If provided, any genotype representing fewer than this fraction of mapped reads will be discarded.
--minPctForLineageFiltering	0.01	This is part of the filtering strategy for ambiguous hits. If provided, references will be grouped by lineage/allotype (based on the supplied file). Within each group any hit sets below this threshold will be ignored. Of the remaining, if any alleles are present across all groups, only those hits will be retained.
--minPctForRef	0.01	This is part of the filtering strategy for ambiguous hits. If provided, any reference with fewer than this fraction of reads mapping to it (of the total reads mapping) will be discarded.
--minReadCountForExport	5	This is part of the filtering strategy for ambiguous hits. If provided, any genotype with fewer than the provided number of reads will be discarded.
--minReadCountForRef	5	This is part of the filtering strategy for ambiguous hits. If provided, any reference with fewer than the provided number of reads mapping to it will be discarded.
--mismatchesTolerated -mm	0	The maximum number of mismatches tolerated for alignment. If a given read has multiple alignments, those with the fewest mismatches will be kept (irrespective of length).
--reference -R	null	Reference sequence
--referenceToLineageFile	null	This is a simple tab-delimited file, no header, with two columns. The first is the reference name, identical to that provided in the FASTA/BAM. The second column is the lineage/allotype of this sequence. This is used for grouping purposes and filtering.
--requireValidPair -rvp	false	If true, only reads with a valid pair to the same reference will be considered
--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.
--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	null	Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--tmp-dir	null	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
--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] []

--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] []

--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 [ [ -∞ ∞ ] ]

--gcs-project-for-requester-pays / NA

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
INTERSECTION

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 / NA

If non-zero, partitions VCF output into shards, each containing up to the given number of records.

int 0 [ [ 0 ∞ ] ]

--minAlignmentLength / -minAlignmentLength

Alignments shorted than this value will be discarded.

Integer 40 [ [ 0 ∞ ] ]

--minMappingQuality / -mmq

Minimum mapping quality of reads to count towards depth.

Integer 0 [ [ -∞ ∞ ] ]

--minPctForExport / -minPctForExport

If provided, any genotype representing fewer than this fraction of mapped reads will be discarded.

Double 0.0 [ [ 0 1 ] ]

--minPctForLineageFiltering / -minPctForLineageFiltering

This is part of the filtering strategy for ambiguous hits. If provided, references will be grouped by lineage/allotype (based on the supplied file). Within each group any hit sets below this threshold will be ignored. Of the remaining, if any alleles are present across all groups, only those hits will be retained.

Double 0.01 [ [ 0 1 ] ]

--minPctForRef / -minPctForRef

This is part of the filtering strategy for ambiguous hits. If provided, any reference with fewer than this fraction of reads mapping to it (of the total reads mapping) will be discarded.

Double 0.01 [ [ 0 1 ] ]

--minReadCountForExport / -minReadCountForExport

This is part of the filtering strategy for ambiguous hits. If provided, any genotype with fewer than the provided number of reads will be discarded.

Integer 5 [ [ 0 ∞ ] ]

--minReadCountForRef / -minReadCountForRef

This is part of the filtering strategy for ambiguous hits. If provided, any reference with fewer than the provided number of reads mapping to it will be discarded.

Integer 5 [ [ 0 ∞ ] ]

--mismatchesTolerated / -mm

The maximum number of mismatches tolerated for alignment. If a given read has multiple alignments, those with the fewest mismatches will be kept (irrespective of length).

Integer 0 [ [ 0 ∞ ] ]

--output / -O

Prefix for output files

R String 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[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

--referenceToLineageFile / -referenceToLineageFile

This is a simple tab-delimited file, no header, with two columns. The first is the reference name, identical to that provided in the FASTA/BAM. The second column is the lineage/allotype of this sequence. This is used for grouping purposes and filtering.

File null