Step 2: Aligns reads to the microbe reference

Category Metagenomics

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Overview

Align reads to a microbe reference using BWA-MEM and Spark. Second step in the PathSeq pipeline.

See PathSeqPipelineSpark for an overview of the PathSeq pipeline.

This is a specialized version of BwaSpark designed for the PathSeq pipeline. The main difference is that alignments with SAM bit flag 0x100 or 0x800 (indicating secondary or supplementary alignment) are omitted in the output.

Inputs

• Unaligned queryname-sorted BAM file containing only paired reads (paired-end reads with mates)
• Unaligned BAM file containing only unpaired reads (paired-end reads without mates and/or single-end reads)
• *Microbe reference BWA-MEM index image generated using BwaMemIndexImageCreator
• *Indexed microbe reference dictionary (fasta file NOT required)

*A standard microbe reference is available in the GATK Resource Bundle.

Output

• Aligned BAM file containing the paired reads (paired-end reads with mates)
• Aligned BAM file containing the unpaired reads (paired-end reads without mates and/or single-end reads)

Usage example

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.

Local mode:

 gatk PathSeqBwaSpark  \
   --paired-input input_reads_paired.bam \
   --unpaired-input input_reads_unpaired.bam \
   --paired-output output_reads_paired.bam \
   --unpaired-output output_reads_unpaired.bam \
   --microbe-bwa-image reference.img \
   --microbe-dict reference.dict
 

Spark cluster on Google Cloud DataProc with 6 32-core / 208GB memory worker nodes:

 gatk PathSeqBwaSpark  \
   --paired-input gs://my-gcs-bucket/input_reads_paired.bam \
   --unpaired-input gs://my-gcs-bucket/input_reads_unpaired.bam \
   --paired-output gs://my-gcs-bucket/output_reads_paired.bam \
   --unpaired-output gs://my-gcs-bucket/output_reads_unpaired.bam \
   --microbe-bwa-image /references/reference.img \
   --microbe-dict hdfs://my-cluster-m:8020//references/reference.dict \
   --bam-partition-size 4000000 \
   -- \
   --sparkRunner GCS \
   --cluster my_cluster \
   --driver-memory 8G \
   --executor-memory 32G \
   --num-executors 4 \
   --executor-cores 30 \
   --conf spark.executor.memoryOverhead=132000
 

Note that the microbe BWA image must be copied to the same path on every worker node. The microbe FASTA may also be copied to a single path on every worker node or to HDFS.

Notes

For small input BAMs, it is recommended that the user reduce the BAM partition size in order to increase parallelism. Note that insert size is estimated separately for each Spark partition. Consequently partition size and other Spark parameters can affect the output for paired-end alignment.

To minimize output file size, header lines are included only for sequences with at least one alignment.

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Additional Information

Read filters

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

• WellformedReadFilter

PathSeqBwaSpark 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
--microbe-bwa-image		Microbe reference BWA index image file generated using BwaMemIndexImageCreator. If running on a Spark cluster, this must be distributed to local disk on each node.
--microbe-dict		Use the given sequence dictionary as the microbe sequence dictionary. Must be a .dict file.
Optional Tool Arguments
--arguments_file		read one or more arguments files and add them to the command line
--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).
--bwa-score-threshold	30	Minimum score threshold for microbe alignments
--conf		Spark properties to set on the Spark context in the format =
--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-alternate-hits	5000	Maximum number of alternate microbe alignments
--microbe-min-seed-length	19	Minimum BWA-MEM seed length for the microbe alignment
--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.
--output-shard-tmp-dir		when writing a bam, in single sharded mode this directory to write the temporary intermediate output shards, if not specified .parts/ will be used
--paired-input		Input queryname-sorted BAM containing only paired reads
--paired-output		Output BAM containing only paired reads
--program-name		Name of the program running
--reference  -R		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.
--spark-verbosity		Spark verbosity. Overrides --verbosity for Spark-generated logs only. Possible values: {ALL, DEBUG, INFO, WARN, ERROR, FATAL, OFF, TRACE}
--unpaired-input		Input BAM containing only unpaired reads
--unpaired-output		Output BAM containing only unpaired reads
--use-nio	false	Whether to use NIO or the Hadoop filesystem (default) for reading files. (Note that the Hadoop filesystem is always used for writing files.)
--version	false	display the version number for this tool
Optional Common Arguments
--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 index when writing a coordinate-sorted BAM file.
--create-output-bam-splitting-index	true	If true, create a BAM splitting index (SBI) when writing a coordinate-sorted BAM file.
--create-output-variant-index  -OVI	true	If true, create a VCF index when writing a coordinate-sorted VCF file.
--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
--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.
--splitting-index-granularity	4096	Granularity to use when writing a splitting index, one entry will be put into the index every n reads where n is this granularity value. Smaller granularity results in a larger index with more available split points.
--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
--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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--add-output-vcf-command-line / -add-output-vcf-command-line

If true, adds a command line header line to created VCF files.

boolean  true

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--arguments_file

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

List[File]  []

---

--bam-partition-size

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  [ [ -∞  ∞ ] ]

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--bwa-score-threshold

Minimum score threshold for microbe alignments
This parameter controls the minimum quality of the BWA alignments for the output.

int  30  [ [ 0  ∞ ] ]

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--conf

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

List[String]  []

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

If true, create a BAM index when writing a coordinate-sorted BAM file.

boolean  true

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--create-output-bam-splitting-index

If true, create a BAM splitting index (SBI) when writing a coordinate-sorted BAM file.

boolean  true

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

If true, create a VCF index when writing a coordinate-sorted VCF file.

boolean  true

---

--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

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--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

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--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]  []

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--gatk-config-file

A configuration file to use with the GATK.

String  null

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--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  ""

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--help / -h

display the help message

boolean  false

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--input / -I

BAM/SAM/CRAM file containing reads

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  [ [ -∞  ∞ ] ]

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--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

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--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  [ [ -∞  ∞ ] ]

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--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

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--intervals / -L

One or more genomic intervals over which to operate

List[String]  []

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--max-alternate-hits

Maximum number of alternate microbe alignments
The maximum number of alternate alignments for each read, i.e. the alignments appearing in the XA tag.

int  5000  [ [ 0  ∞ ] ]

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--microbe-bwa-image

Microbe reference BWA index image file generated using BwaMemIndexImageCreator. If running on a Spark cluster, this must be distributed to local disk on each node.

R String  null

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--microbe-dict

Use the given sequence dictionary as the microbe sequence dictionary. Must be a .dict file.

R String  null

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--microbe-min-seed-length

Minimum BWA-MEM seed length for the microbe alignment
This parameter controls the sensitivity of the BWA-MEM aligner. Smaller values result in more alignments at the expense of computation time.

int  19  [ [ 1  [ 11  ∞ ] ]

---

--num-reducers

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-shard-tmp-dir

when writing a bam, in single sharded mode this directory to write the temporary intermediate output shards, if not specified .parts/ will be used

Exclusion: This argument cannot be used at the same time as sharded-output.

String  null

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--paired-input

Input queryname-sorted BAM containing only paired reads

String  null

---

--paired-output

Output BAM containing only paired reads

String  null

---

--program-name

Name of the program running

String  null

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--QUIET

Whether to suppress job-summary info on System.err.

Boolean  false

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--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]  []

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--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

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--reference / -R

Reference sequence

GATKPath  null

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--sharded-output

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

Exclusion: This argument cannot be used at the same time as output-shard-tmp-dir.

boolean  false

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--showHidden / -showHidden

display hidden arguments

boolean  false

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--spark-master

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

String  local[*]

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--spark-verbosity

Spark verbosity. Overrides --verbosity for Spark-generated logs only. Possible values: {ALL, DEBUG, INFO, WARN, ERROR, FATAL, OFF, TRACE}

String  null

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--splitting-index-granularity

Granularity to use when writing a splitting index, one entry will be put into the index every n reads where n is this granularity value. Smaller granularity results in a larger index with more available split points.

long  4096  [ [ 1  ∞ ] ]

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--tmp-dir

Temp directory to use.

GATKPath  null

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--unpaired-input

Input BAM containing only unpaired reads

String  null

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--unpaired-output

Output BAM containing only unpaired reads

String  null

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

Whether to use the JdkDeflater (as opposed to IntelDeflater)

boolean  false

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

Whether to use the JdkInflater (as opposed to IntelInflater)

boolean  false

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--use-nio

Whether to use NIO or the Hadoop filesystem (default) for reading files. (Note that the Hadoop filesystem is always used for writing files.)

boolean  false

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--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

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--version

display the version number for this tool

boolean  false

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GATK version 4.3.0.0 built at Wed, 12 Oct 2022 21:04:44 -0400.