Step 1: Filters low quality, low complexity, duplicate, and host reads

Category Metagenomics

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Overview

Filters low complexity, low quality, duplicate, and host reads. First step in the PathSeq pipeline.

See PathSeqPipelineSpark for an overview of the PathSeq pipeline.

Methods

The tool works by filtering out reads in a series of stages:

1. Remove secondary and supplementary alignments
2. If the input reads are aligned to a host reference, remove mapped reads with sufficient alignment identity
3. Trim adapter sequences
4. Mask sequences with excessive A/T or G/C content
5. Mask repetitive sequences (see Liebert et al. 2006)
6. Hard clip read ends using base qualities
7. Remove reads that are too short or were trimmed excessively
8. Mask low-quality bases
9. Remove reads with too many masked bases
10. Remove reads containing one or more host k-mers
11. Remove reads with sufficient alignment identity to the host reference
12. Remove exact duplicate reads

Input

• BAM containing input reads (either unaligned or aligned to a host reference)
• *Host k-mer file generated using PathSeqBuildKmers
• *Host BWA-MEM index image generated using BwaMemIndexImageCreator

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

Output

• BAM containing high quality, non-host paired reads (paired-end reads with both mates)
• BAM containing high quality, non-host unpaired reads (single-end reads and/or paired-end reads without mates)

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 PathSeqFilterSpark  \
   --input input_reads.bam \
   --paired-output output_reads_paired.bam \
   --unpaired-output output_reads_unpaired.bam \
   --min-clipped-read-length 60 \
   --kmer-file host_kmers.bfi \
   --filter-bwa-image host_reference.img \
   --filter-metrics metrics.txt \
   --bam-partition-size 4000000
 

Spark cluster on Google Cloud DataProc with 4 16-core / 100GB memory worker nodes:

 gatk PathSeqFilterSpark  \
   --input gs://my-gcs-bucket/input_reads.bam \
   --paired-output gs://my-gcs-bucket/output_reads_paired.bam \
   --unpaired-output gs://my-gcs-bucket/output_reads_unpaired.bam \
   --min-clipped-read-length 60 \
   --kmer-file hdfs://my-cluster-m:8020//references/host_kmers.bfi \
   --filter-bwa-image /references/host_reference.img \
   --filter-metrics gs://my-gcs-bucket/metrics.txt \
   --bam-partition-size 4000000 \
   -- \
   --sparkRunner GCS \
   --cluster my_cluster \
   --driver-memory 8G \
   --executor-memory 32G \
   --num-executors 4 \
   --executor-cores 15 \
   --conf spark.yarn.executor.memoryOverhead=32000
 

Note that the host BWA image must be copied to the same path on every worker node. The host k-mer file may also be copied to a single path on every worker node or to HDFS.

Notes

The input BAM may be unaligned (a uBAM) or aligned to a host reference (e.g. hg38). If it is aligned then --is-host-aligned should be enabled. This will substantially increase performance, as host reads can then be immediately subtracted prior to quality filtering and host alignment.

References

1. Liebert, M. A. et al. (2006). A Fast and Symmetric DUST Implementation to Mask Low-Complexity DNA Sequences. J. Comput. Biol., 13, 1028-1040.

PathSeqFilterSpark 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
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).
--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!
--dust-mask-quality	2	Base quality to assign low-complexity bases
--dust-t	20.0	DUST algorithm score threshold
--dust-window	64	DUST algorithm window size
--filter-bwa-image  -FI	null	The BWA image file of the host reference. This must be distributed to local disk on each node.
--filter-bwa-seed-length	19	Minimum seed length for the host BWA alignment.
--filter-duplicates	true	Filter duplicate reads
--filter-metrics  -FM	null	Log counts of filtered reads to this file
--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.
--help  -h	false	display the help message
--host-kmer-thresh	1	Host kmer count threshold.
--host-min-identity	30	Host alignment identity score threshold, in bp
--interval-merging-rule  -imr	ALL	Interval merging rule for abutting intervals
--intervals  -L	[]	One or more genomic intervals over which to operate
--is-host-aligned	false	Set if the input BAM is aligned to the host
--kmer-file  -K	null	Path to host k-mer file generated with PathSeqBuildKmers. K-mer filtering is skipped if this is not specified.
--max-adapter-mismatches	1	Maximum number of mismatches for adapter trimming
--max-masked-bases	2	Max allowable number of masked bases per read
--min-adapter-length	12	Minimum length of adapter sequence to trim
--min-base-quality	15	Bases below this call quality will be masked with 'N'
--min-clipped-read-length	31	Minimum length of reads after quality trimming
--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	null	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-output  -PO	null	Output BAM containing only paired reads
--program-name	null	Name of the program running
--quality-threshold	15	Quality score trimmer threshold
--reference  -R	null	Reference sequence
--sharded-output	false	For tools that write an output, write the output in multiple pieces (shards)
--skip-quality-filters	false	Skip low-quality and low-complexity read filtering
--spark-master	local[*]	URL of the Spark Master to submit jobs to when using the Spark pipeline runner.
--unpaired-output  -UO	null	Output BAM containing only unpaired reads
--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.
--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	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
--filter-reads-per-partition	200000	Estimated reads per partition after quality, kmer, and BWA filtering
--showHidden	false	display hidden arguments
--skip-pre-bwa-repartition	false	Skip pre-BWA repartition. Set to true for inputs with a high proportion of microbial reads that are not host coordinate-sorted.

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 / NA

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

List[File]  []

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--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  [ [ -∞  ∞ ] ]

---

--conf / -conf

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

List[String]  []

---

--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

---

--dust-mask-quality / -dust-mask-quality

Base quality to assign low-complexity bases

int  2  [ [ -∞  ∞ ] ]

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--dust-t / -dust-t

DUST algorithm score threshold
Controls the stringency of low-complexity filtering.

double  20.0  [ [ -∞  ∞ ] ]

---

--dust-window / -dust-window

DUST algorithm window size

int  64  [ [ -∞  ∞ ] ]

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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]  []

---

--filter-bwa-image / -FI

The BWA image file of the host reference. This must be distributed to local disk on each node.
This file should be generated using BwaMemIndexImageCreator.

String  null

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--filter-bwa-seed-length / -filter-bwa-seed-length

Minimum seed length for the host BWA alignment.
Controls the sensitivity of BWA alignment to the host reference. Shorter seed lengths will enhance detection of host reads during the subtraction phase but will also increase run time.

int  19  [ [ 1  [ 11  ∞ ] ]

---

--filter-duplicates / -filter-duplicates

Filter duplicate reads
If true, then for any two reads with identical sequences (or identical to the other's reverse complement), one will be filtered.

boolean  true

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--filter-metrics / -FM

Log counts of filtered reads to this file
If specified, records the number of reads remaining after each of the following steps:

• Pre-aligned host read filtering
• Low-quality and low-complexity sequence filtering
• Host read subtraction
• Read deduplication

It also provides the following:

• Number of low-quality and low-complexity reads removed
• Number of host reads removed
• Number of duplicate reads removed
• Final number of reads
• Final number of paired reads
• Final number of unpaired reads

Note that using this option may substantially increase runtime.

String  null

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--filter-reads-per-partition / -filter-reads-per-partition

Estimated reads per partition after quality, kmer, and BWA filtering
This is a parameter for fine-tuning memory performance. Lower values may result in less memory usage but possibly at the expense of greater computation time.

int  200000  [ [ 1  ∞ ] ]

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--gatk-config-file / NA

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 / NA

Project to bill when accessing "requester pays" buckets. If unset, these buckets cannot be accessed.

String  ""

---

--help / -h

display the help message

boolean  false

---

--host-kmer-thresh / -host-kmer-thresh

Host kmer count threshold.
Controls the stringency of read filtering based on host k-mer matching. Reads with at least this many matching k-mers in the host reference will be filtered.

int  1  [ [ 1  ∞ ] ]

---

--host-min-identity / -host-min-identity

Host alignment identity score threshold, in bp
Controls the stringency of read filtering based on alignment to the host reference. The identity score is defined as the number of matching bases less the number of deletions in the alignment.

int  30  [ [ 1  ∞ ] ]

---

--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  [ [ -∞  ∞ ] ]

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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

---

--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]  []

---

--is-host-aligned / -is-host-aligned

Set if the input BAM is aligned to the host
PathSeq will rapidly filter the reads if they are aligned to a host reference, thus reducing run time.

boolean  false

---

--kmer-file / -K

Path to host k-mer file generated with PathSeqBuildKmers. K-mer filtering is skipped if this is not specified.

String  null

---

--max-adapter-mismatches / -max-adapter-mismatches

Maximum number of mismatches for adapter trimming

int  1  [ [ 0  ∞ ] ]

---

--max-masked-bases / -max-masked-bases

Max allowable number of masked bases per read
This is the threshold for filtering reads based on the number of 'N' values present in the sequence. Note that the low-complexity DUST filter and quality filter mask using 'N' bases. Therefore, this parameter is the threshold for the sum of:

• The number of N's in the original input read
• The number of low-quality base calls
• The number of low-complexity bases

int  2  [ [ 0  ∞ ] ]

---

--min-adapter-length / -min-adapter-length

Minimum length of adapter sequence to trim
Adapter trimming will require a match of at least this length to a known adapter.

int  12  [ [ 1  ∞ ] ]

---

--min-base-quality / -min-base-quality

Bases below this call quality will be masked with 'N'

int  15  [ [ 1  ∞ ] ]

---

--min-clipped-read-length / -min-clipped-read-length

Minimum length of reads after quality trimming
Reads are trimmed based on base call quality and low-complexity content. Decreasing the value will enhance pathogen detection (higher sensitivity) but also result in undesired false positives and ambiguous microbe alignments (lower specificity).

int  31  [ [ 1  [ 31  ∞ ] ]

---

--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-shard-tmp-dir / NA

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-output / -PO

Output BAM containing only paired reads

String  null

---

--program-name / NA

Name of the program running

String  null

---

--quality-threshold / -quality-threshold

Quality score trimmer threshold
Controls the stingency of base call quality-based read trimming. Higher values result in more trimming.

int  15  [ [ 1  ∞ ] ]

---

--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-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

---

--sharded-output / NA

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

---

--skip-pre-bwa-repartition / -skip-pre-bwa-repartition

Skip pre-BWA repartition. Set to true for inputs with a high proportion of microbial reads that are not host coordinate-sorted.

Advanced optimization option that should be used only in the case of inputs with a high proportion of microbial reads that are not host-aligned/coordinate-sorted.

In the filter tool, the input reads are initially divided up into smaller partitions (default size is usually the size of one HDFS block, or ~64MB) that Spark works on in parallel. In samples with a low proportion of microbial reads (e.g. < 1%), the steps leading up to the host BWA alignment will whittle these partitions down to a small fraction of their original size. At that point, the distribution of reads across the partitions may be unbalanced.

For example, say the input is 256MB and Spark splits this into 4 even partitions. It is possible that, after running through the quality filters and host kmer search, there are 5% remaining in partition #1, 8% in partition #2, 2% in partition #3, and 20% in partition #4. Thus there is an imbalance of work across the partitions. To correct this, a "reparitioning" is invoked that distributes the reads evenly. Note this is especially important for host-aligned, coordinate-sorted inputs, in which unmapped reads would be concentrated in the last partitions.

If, however, the proportion of microbial reads is higher, say 30%, then the partitions are generally more balanced (except for in the aforementioned coordinate-sorted case). In this case, the time spent doing the repartitioning is usually greater than the time saved by rebalancing, and this option should be invoked.

boolean  false

---

--skip-quality-filters / -skip-quality-filters

Skip low-quality and low-complexity read filtering

boolean  false

---

--spark-master / NA

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

String  local[*]

---

--tmp-dir / NA

Temp directory to use.

String  null

---

--unpaired-output / -UO

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

---

--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

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GATK version 4.0.12.0 built at 23-45-2019 03:45:45.