Step 2: Aligns reads to the microbe reference
Category Metagenomics
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.
Additional Information
Read filters
This Read Filter is automatically applied to the data by the Engine before processing by PathSeqBwaSpark.
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.
--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] []
--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 [ [ -∞ ∞ ] ]
--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 ∞ ] ]
--conf
Spark properties to set on the Spark context in the format =
List[String] []
--create-output-bam-index / -OBI
If true, create a BAM index when writing a coordinate-sorted BAM file.
boolean true
--create-output-bam-splitting-index
If true, create a BAM splitting index (SBI) when writing a coordinate-sorted BAM file.
boolean true
--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
--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] []
--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
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] []
--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 ∞ ] ]
--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
--microbe-dict
Use the given sequence dictionary as the microbe sequence dictionary. Must be a .dict file.
R String null
--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
--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
--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
GATKPath null
--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
--showHidden / -showHidden
display hidden arguments
boolean false
--spark-master
URL of the Spark Master to submit jobs to when using the Spark pipeline runner.
String local[*]
--spark-verbosity
Spark verbosity. Overrides --verbosity for Spark-generated logs only. Possible values: {ALL, DEBUG, INFO, WARN, ERROR, FATAL, OFF, TRACE}
String null
--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 ∞ ] ]
--tmp-dir
Temp directory to use.
GATKPath null
--unpaired-input
Input BAM containing only unpaired reads
String null
--unpaired-output
Output BAM containing only unpaired reads
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
--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
--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
GATK version 4.2.4.0-SNAPSHOT built at Thu, 16 Dec 2021 11:57:48 -0800.
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