(Internal) Produces local assemblies of genomic regions that may harbor structural variants

Category Structural Variant Discovery

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Overview

(Internal) Produces local assemblies of genomic regions that may harbor structural variants

This tool is used in development and should not be of interest to most researchers. It packages the identification of genomic regions that might contain structural variation and the generation of local assemblies of these regions as a separate tool, independent of the calling of structural variations from these assemblies. Most researchers will run StructuralVariationDiscoveryPipelineSpark, which both generates local assemblies of interesting genomic regions, and then calls structural variants from these assemblies.

This tool identifies genomic regions that may harbor structural variants by integrating evidence from split reads, discordant read pairs, template-length anomalies, and copy-number variation. It then prepares local assemblies of these regions for structural variant calling. In addition to the reads that align to these regions, reads sharing kmers (fixed-length subsequences) with the reads aligned in these regions are extracted to produce the local assemblies.

The local assemblies are done with FermiLite, and the assembled contigs are aligned to reference with BWA-MEM.

The output is a file of aligned contigs from local assemblies to be used in calling structural variants.

Inputs

• A SAM/BAM/CRAM file of paired-end, aligned and coordinate-sorted reads.
• A BWA index image for the reference. You can use BwaMemIndexImageCreator to create the index image file.
• A list of ubiquitous kmers to ignore. You can use FindBadGenomicGenomicKmersSpark to create the list of kmers to ignore.

Output

• A file of aligned contigs.

Usage example

   gatk FindBreakpointEvidenceSpark \
     -I input_reads.bam \
     --aligner-index-image reference.img \
     --kmers-to-ignore ignored_kmers.txt \
     -O assemblies.sam
 

This tool can be run without explicitly specifying Spark options. That is to say, the given example command without Spark options will run locally. See Tutorial#10060 for an example of how to set up and run a Spark tool on a cloud Spark cluster.

Caveats

Expected input is a paired-end, coordinate-sorted BAM with around 30x coverage. Coverage much lower than that probably won't work well.

FindBreakpointEvidenceSpark 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
--aligner-index-image	null	bwa-mem index image file
--input  -I	[]	BAM/SAM/CRAM file containing reads
--kmers-to-ignore	null	file containing ubiquitous kmer list. see FindBadGenomicKmersSpark to generate it.
--output  -O	null	sam file for aligned contigs
Optional Tool Arguments
--adapter-sequence	null	Adapter sequence.
--allowed-short-fragment-overhang	10	Proper pairs have the positive strand read upstream of the negative strand read, but we allow this much slop for short fragments.
--arguments_file	[]	read one or more arguments files and add them to the command line
--assembled-contigs-output-order  -sort	coordinate	sorting order to be used for the output assembly alignments SAM/BAM file
--assembly-to-mapped-size-ratio-guess	7	Guess at the ratio of reads in the final assembly to the number reads mapped to the interval.
--bam-partition-size	0	maximum number of bytes to read from a file into each partition of reads. Setting this higher will result in fewer partitions. Note that this will not be equal to the size of the partition in memory. Defaults to 0, which uses the default split size (determined by the Hadoop input format, typically the size of one HDFS block).
--breakpoint-evidence-dir	null	directory for evidence output
--breakpoint-intervals	null	file for breakpoint intervals output
--cleaner-max-copy-number	4	KmerCleaner maximum copy number (not count, but copy number) for a kmer. Kmers observed too frequently are probably mismapped or ubiquitous.
--cleaner-max-intervals	3	KmerCleaner maximum number of intervals for a localizing kmer. If a kmer occurs in too many intervals, it isn't sufficiently local.
--cleaner-min-kmer-count	4	KmerCleaner minimum kmer count for a localizing kmer. If we see it less often than this many times, we're guessing it's erroneous.
--conf	[]	spark properties to set on the spark context in the format =
--cross-contigs-to-ignore	null	file containing alt contig names that will be ignored when looking for inter-contig pairs
--disable-sequence-dictionary-validation	false	If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
--exclusion-interval-padding	0	Exclusion interval padding.
--exclusion-intervals	null	file of reference intervals to exclude
--external-evidence	null	external evidence input file
--external-evidence-uncertainty	150	Uncertainty in location of external evidence.
--external-evidence-weight	10	Weight to give external evidence.
--fastq-dir	null	output dir for assembled fastqs
--gcs-max-retries  -gcs-retries	20	If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
--help  -h	false	display the help message
--high-coverage-intervals	null	file for high-coverage intervals output
--high-depth-coverage-factor	3	We filter out contiguous regions of the genome that have coverage of at least high-depth-coverage-factor * avg-coverage and a peak coverage of high-depth-coverage-peak-factor * avg-coverage, because the reads mapped to those regions tend to be non-local and high depth prevents accurate assembly.
--high-depth-coverage-peak-factor	7	We filter out contiguous regions of the genome that have coverage of at least high-depth-coverage-factor * avg-coverage and a peak coverage of high-depth-coverage-peak-factor * avg-coverage, because the reads mapped to those regions tend to be non-local and high depth prevents accurate assembly.
--include-mapping-location	true	Include read mapping location in FASTQ files.
--interval-merging-rule  -imr	ALL	Interval merging rule for abutting intervals
--interval-only-assembly	false	Don't look for extra reads mapped outside the interval.
--intervals  -L	[]	One or more genomic intervals over which to operate
--k-size	51	Kmer size.
--kmer-intervals	null	file for kmer intervals output
--kmer-max-dust-score	49	Maximum kmer DUST score.
--max-fastq-size	3000000	Maximum total bases in FASTQs that can be assembled.
--max-tracked-fragment-length	2000	Largest fragment size that will be explicitly counted in determining fragment size statistics.
--min-coherent-evidence-count	7	Minimum weight of the evidence that shares a distal target locus to validate the evidence.
--min-evidence-count	15	Minimum weight of the corroborating read evidence to validate some single piece of evidence.
--min-evidence-mapq	20	The minimum mapping quality for reads used to gather evidence of breakpoints.
--min-evidence-match-length	45	The minimum length of the matched portion of an interesting alignment. Reads that don't match at least this many reference bases won't be used in gathering evidence.
--min-kmers-per-interval	5	Minimum number of localizing kmers in a valid interval.
--num-reducers	0	For tools that shuffle data or write an output, sets the number of reducers. Defaults to 0, which gives one partition per 10MB of input.
--program-name	null	Name of the program running
--qname-intervals-for-assembly	null	file for mapped qname intervals output
--qname-intervals-mapped	null	file for mapped qname intervals output
--read-metadata	null	output file for read metadata
--reference  -R	null	Reference sequence
--sharded-output	false	For tools that write an output, write the output in multiple pieces (shards)
--spark-master	local[*]	URL of the Spark Master to submit jobs to when using the Spark pipeline runner.
--target-link-file	null	output file for non-assembled breakpoints in bedpe format
--unfiltered-breakpoint-evidence-dir	null	directory for evidence output
--version	false	display the version number for this tool
--write-gfas	false	Write GFA representation of assemblies in fastq-dir.
Optional Common Arguments
--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
--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.
--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
--expand-assembly-graph	true	Traverse assembly graph and produce contigs for all paths.
--pop-variant-bubbles	false	Aggressively simplify local assemblies, ignoring small variants.
--remove-shadowed-contigs	true	Simplify local assemblies by removing contigs shadowed by similar contigs.
--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.

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--adapter-sequence / NA

Adapter sequence.

String  null

---

--aligner-index-image / NA

bwa-mem index image file

R String  null

---

--allowed-short-fragment-overhang / NA

Proper pairs have the positive strand read upstream of the negative strand read, but we allow this much slop for short fragments.

int  10  [ [ -∞  ∞ ] ]

---

--arguments_file / NA

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

List[File]  []

---

--assembled-contigs-output-order / -sort

sorting order to be used for the output assembly alignments SAM/BAM file

The --assembled-contigs-output-order argument is an enumerated type (SortOrder), which can have one of the following values:

unsorted

queryname

coordinate

duplicate

unknown

SortOrder  coordinate

---

--assembly-to-mapped-size-ratio-guess / NA

Guess at the ratio of reads in the final assembly to the number reads mapped to the interval.

int  7  [ [ -∞  ∞ ] ]

---

--bam-partition-size / NA

maximum number of bytes to read from a file into each partition of reads. Setting this higher will result in fewer partitions. Note that this will not be equal to the size of the partition in memory. Defaults to 0, which uses the default split size (determined by the Hadoop input format, typically the size of one HDFS block).

long  0  [ [ -∞  ∞ ] ]

---

--breakpoint-evidence-dir / NA

directory for evidence output

String  null

---

--breakpoint-intervals / NA

file for breakpoint intervals output

String  null

---

--cleaner-max-copy-number / NA

KmerCleaner maximum copy number (not count, but copy number) for a kmer. Kmers observed too frequently are probably mismapped or ubiquitous.

int  4  [ [ -∞  ∞ ] ]

---

--cleaner-max-intervals / NA

KmerCleaner maximum number of intervals for a localizing kmer. If a kmer occurs in too many intervals, it isn't sufficiently local.

int  3  [ [ -∞  ∞ ] ]

---

--cleaner-min-kmer-count / NA

KmerCleaner minimum kmer count for a localizing kmer. If we see it less often than this many times, we're guessing it's erroneous.

int  4  [ [ -∞  ∞ ] ]

---

--conf / -conf

spark properties to set on the spark context in the format =

List[String]  []

---

--cross-contigs-to-ignore / NA

file containing alt contig names that will be ignored when looking for inter-contig pairs
This is a path to a text file of contig names (one per line) that will be ignored when looking for inter-contig pairs.

String  null

---

--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).

List[String]  []

---

--exclusion-interval-padding / NA

Exclusion interval padding.

int  0  [ [ -∞  ∞ ] ]

---

--exclusion-intervals / NA

file of reference intervals to exclude
This is a file that calls out the coordinates of intervals in the reference assembly to exclude from consideration when calling putative breakpoints. Each line is a tab-delimited interval with 1-based inclusive coordinates like this: chr1 124535434 142535434

String  null

---

--expand-assembly-graph / NA

Traverse assembly graph and produce contigs for all paths.

boolean  true

---

--external-evidence / NA

external evidence input file

String  null

---

--external-evidence-uncertainty / NA

Uncertainty in location of external evidence.

int  150  [ [ -∞  ∞ ] ]

---

--external-evidence-weight / NA

Weight to give external evidence.

int  10  [ [ -∞  ∞ ] ]

---

--fastq-dir / NA

output dir for assembled fastqs

String  null

---

--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  [ [ -∞  ∞ ] ]

---

--help / -h

display the help message

boolean  false

---

--high-coverage-intervals / NA

file for high-coverage intervals output

String  null

---

--high-depth-coverage-factor / NA

We filter out contiguous regions of the genome that have coverage of at least high-depth-coverage-factor * avg-coverage and a peak coverage of high-depth-coverage-peak-factor * avg-coverage, because the reads mapped to those regions tend to be non-local and high depth prevents accurate assembly.

int  3  [ [ -∞  ∞ ] ]

---

--high-depth-coverage-peak-factor / NA

We filter out contiguous regions of the genome that have coverage of at least high-depth-coverage-factor * avg-coverage and a peak coverage of high-depth-coverage-peak-factor * avg-coverage, because the reads mapped to those regions tend to be non-local and high depth prevents accurate assembly.

int  7  [ [ -∞  ∞ ] ]

---

--include-mapping-location / NA

Include read mapping location in FASTQ files.

boolean  true

---

--input / -I

BAM/SAM/CRAM file containing reads

R List[String]  []

---

--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-only-assembly / NA

Don't look for extra reads mapped outside the interval.

boolean  false

---

--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]  []

---

--k-size / NA

Kmer size.

int  51  [ [ -∞  ∞ ] ]

---

--kmer-intervals / NA

file for kmer intervals output

String  null

---

--kmer-max-dust-score / NA

Maximum kmer DUST score.

int  49  [ [ -∞  ∞ ] ]

---

--kmers-to-ignore / NA

file containing ubiquitous kmer list. see FindBadGenomicKmersSpark to generate it.
This is a path to a file of kmers that appear too frequently in the reference to be usable as probes to localize reads. We don't calculate it here, because it depends only on the reference. The program FindBadGenomicKmersSpark can produce such a list for you.

R String  null

---

--max-fastq-size / NA

Maximum total bases in FASTQs that can be assembled.

int  3000000  [ [ -∞  ∞ ] ]

---

--max-tracked-fragment-length / NA

Largest fragment size that will be explicitly counted in determining fragment size statistics.

int  2000  [ [ -∞  ∞ ] ]

---

--min-coherent-evidence-count / NA

Minimum weight of the evidence that shares a distal target locus to validate the evidence.

int  7  [ [ -∞  ∞ ] ]

---

--min-evidence-count / NA

Minimum weight of the corroborating read evidence to validate some single piece of evidence.

int  15  [ [ -∞  ∞ ] ]

---

--min-evidence-mapq / NA

The minimum mapping quality for reads used to gather evidence of breakpoints.

int  20  [ [ -∞  ∞ ] ]

---

--min-evidence-match-length / NA

The minimum length of the matched portion of an interesting alignment. Reads that don't match at least this many reference bases won't be used in gathering evidence.

int  45  [ [ -∞  ∞ ] ]

---

--min-kmers-per-interval / NA

Minimum number of localizing kmers in a valid interval.

int  5  [ [ -∞  ∞ ] ]

---

--num-reducers / NA

For tools that shuffle data or write an output, sets the number of reducers. Defaults to 0, which gives one partition per 10MB of input.

int  0  [ [ -∞  ∞ ] ]

---

--output / -O

sam file for aligned contigs

R String  null

---

--pop-variant-bubbles / NA

Aggressively simplify local assemblies, ignoring small variants.

boolean  false

---

--program-name / NA

Name of the program running

String  null

---

--qname-intervals-for-assembly / NA

file for mapped qname intervals output

String  null

---

--qname-intervals-mapped / NA

file for mapped qname intervals output

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[String]  []

---

--read-metadata / NA

output file for read metadata

String  null

---

--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

String  null

---

--remove-shadowed-contigs / NA

Simplify local assemblies by removing contigs shadowed by similar contigs.

boolean  true

---

--sharded-output / NA

For tools that write an output, write the output in multiple pieces (shards)

boolean  false

---

--showHidden / -showHidden

display hidden arguments

boolean  false

---

--spark-master / NA

URL of the Spark Master to submit jobs to when using the Spark pipeline runner.

String  local[*]

---

--target-link-file / NA

output file for non-assembled breakpoints in bedpe format

String  null

---

--TMP_DIR / NA

Undocumented option

List[File]  []

---

--unfiltered-breakpoint-evidence-dir / NA

directory for evidence output

String  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

---

--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 / NA

display the version number for this tool

boolean  false

---

--write-gfas / NA

Write GFA representation of assemblies in fastq-dir.

boolean  false

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GATK version 4.0.4.0 built at 02-47-2019 02:47:54.