FASTQ AND BAM PROCESSING OVERVIEW

NVIDIA Clara Parabricks Pipelines tools that can process fastq files and refine bam files

Here are the articles in this section:

Generate BAM output given one or more pairs of fastq files. Optionally generate BQSR report.

fq2bam performs the following steps. User can decide to turn-off marking of duplicates. BQSR step is only performed if –knownSites input and –out-recal-file options are provided.

QUICK START

            

            
$ pbrun fq2bam --ref Ref/Homo_sapiens_assembly38.fasta \
--in-fq Data/sample_1.fq.gz Data/sample_2.fq.gz  \
--knownSites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
--out-bam mark_dups_gpu.bam \
--out-recal-file recal_gpu.txt \
--tmp-dir /raid/myrun
        

COMPATIBLE CPU BASED BWA-MEM, GATK4 COMMANDS

The command below is the bwa-0.7.15 and GATK4 counterpart of the Parabricks command above. The output from these commands will generate the exact same results as the output from the above command. Please look at Output Comparison page on how you can compare the results.

            

            
# Run bwa-mem and pipe output to create sorted bam
$ bwa mem -t 32 -K 10000000 -R '@RG\tID:sample_rg1\tLB:lib1\tPL:bar\tSM:sample\tPU:sample_rg1' \
Ref/Homo_sapiens_assembly38.fasta Data/sample_1.fq.gz Data/sample_2.fq.gz | gatk \
SortSam --java-options -Xmx30g --MAX_RECORDS_IN_RAM=5000000 -I=/dev/stdin \
-O=cpu.bam --SORT_ORDER=coordinate --TMP_DIR=/raid/myrun

# Mark Duplicates
$ gatk MarkDuplicates --java-options -Xmx30g -I=cpu.bam -O=mark_dups_cpu.bam \
-M=metrics.txt --TMP_DIR=/raid/myrun

# Generate BQSR Report
$ gatk BaseRecalibrator --java-options -Xmx30g --input mark_dups_cpu.bam --output \
recal_cpu.txt --known-sites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
--reference Ref/Homo_sapiens_assembly38.fasta
        

OPTIONS

--ref

(required) The reference genome in fasta format. We assume that the indexing required to run bwa has been completed by the user.

--in-fq

(required) Pair ended fastq files. These can be in .fastq or .fastq.gz format. You can provide read group information as an optional third argument.

Example 1:

--in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz

Example 2:

--in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1"

This option can be repeated multiple times as well.

Example 1:

--in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz

Example 2:

--in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" \

--in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2"

Either all sets of inputs have read group or none should have it and will be automatically added by the pipeline. For same sample, Read Groups should have same sample name (SM) and different ID and PU.

--in-se-fq

(required) Single ended fastq files. These can be in .fastq or .fastq.gz format. You can provide read group information as an optional third argument.

Example 1:

--in-se-fq sampleX.fastq.gz

Example 2:

--in-se-fq sampleX.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1"

This option can be repeated multiple times as well.

Example 1:

--in-se-fq sampleX_1.fastq.gz --in-se-fq sampleX_2.fastq.gz

Example 2:

--in-se-fq sampleX_1.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" \

--in-se-fq sampleX_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2"

Either all sets of inputs have read group or none should have it and will be automatically added by the pipeline. For the same sample, Read Groups should have the same sample name (SM) and different ID and PU.

--out-bam

(required) Path to the file that will contain BAM output.

--out-recal-file

Path of report file (.txt format) after Base Quality Score Recalibration. This option can be passed in, if –knownSites are provided.

--out-duplicate-metrics

Path of duplicate metrics file after Marking Duplicates.

--mba

Defaults to False.

Run MergeBAMAlignment after alignment.

--in-mba-file

Path to a file containing options for MergeBAMAlignment. Currently supported options are ATTRIBUTES_TO_RETAIN, ATTRIBUTES_TO_REMOVE, ATTRIBUTES_TO_REMOVE, MAX_INSERTIONS_OR_DELETIONS, PRIMARY_ALIGNMENT_STRATEGY, UNMAPPED_READ_STRATEGY, ALIGNER_PROPER_PAIR_FLAGS, UNMAP_CONTAMINANT_READS, and ADD_PG_TAG_TO_READS. Each option must be specified on a new line in the file. Refer to Picard MergeBAMAlignment documentation for details on these options.

--knownSites

Known indel files in .vcf.gz format. These should be compressed vcf files for known SNPs and indels. You can use this option multiple times. If you provide this option, then you must also provide an –out-recal-file.

--interval-file

Path to an interval file for BQSR step with possible formats:

Picard-style (.interval_list or .picard), GATK-style (.list or .intervals), or BED file (.bed). This option can be used multiple times (default: None)

--interval

(-L) Interval strings within which to call BQSR from the input reads. All intervals will have a padding of 100 to get read records and overlapping intervals will be combined. Interval files should be passed using the –interval-file option. This option can be used multiple times. e.g. “-L chr1 -L chr2:10000 -L chr3:20000+ -L chr4:10000-20000” (default: None)

--interval-padding

(-ip) Padding size (in base pairs) to add to each interval you are including (default: None)

--no-markdups

Defaults to False.

Do not mark duplicates, generate bam after co-ordinate sorting.

--bwa-options

Pass supported bwa mem options as one string. Current original bwa mem supported options: -M, -Y, -T .

--markdups-assume-sortorder-queryname

Defaults to False.

Assume the reads are sorted by queryname for Marking Duplicates. This will mark secondary, supplementary and unmapped reads as duplicates as well. This flag will not impact variant calling while increasing processing times.

--optical-duplicate-pixel-distance

The maximum offset between two duplicate clusters in order to consider them optical duplicates. This option is only used if you provide an –out-duplicate-metrics.

--read-group-sm

SM tag for read groups in this run.

--read-group-lb

LB tag for read groups in this run.

--read-group-pl

PL tag for read groups in this run.

--read-group-id-prefix

Prefix for ID and PU tag for read groups in this run. This prefix will be used for all pair of fastq files in this run. The ID and PU tag will consist of this prefix and an identifier which will be unique for a pair of fastq files.

--tmp-dir

Defaults to ..

Full path to the directory where temporary files will be stored.

--num-gpus

Defaults to number of GPUs in the system.

The number of GPUs to be used for this analysis task.

--gpu-devices

Which GPU devices to use for a run. By default, all GPU devices will be used. To set specific GPU devices, enter a comma-separated list of GPU device numbers.

bqsr performs the Base Quality Score Recalibration (BQSR) in a stand alone fashion.

QUICK START

            

            
$ pbrun bqsr --ref Ref/Homo_sapiens_assembly38.fasta \
--in-bam mark_dups_gpu.bam \
--knownSites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
--out-recal-file recal_gpu.txt \
        

COMPATIBLE GATK4 COMMAND

The command below is the GATK4 counterpart of the Parabricks command above. The output from these commands will generate the exact same results as the output from the above command.

            

            
$ gatk BaseRecalibrator --java-options -Xmx30g --input mark_dups_gpu.bam --output \
recal_cpu.txt --known-sites Ref/Homo_sapiens_assembly38.known_indels.vcf.gz \
--reference Ref/Homo_sapiens_assembly38.fasta
        

OPTIONS

--ref

(required) The reference genome in fasta format. We assume that the indexing required to run bwa has been completed by the user.

--in-bam

(required) Path to the input BAM file.

--knownSites

(required) Path to a known indels file. Must be in vcf/vcf.gz format. This option can be used multiple times.

--interval-file

Path to an interval file for BQSR step with possible formats: Picard-style (.interval_list or .picard), GATK-style (.list or .intervals), or BED file (.bed). This option can be used multiple times (default: None)

--interval

(-L) Interval within which to call BQSR from the input reads. All intervals will have a padding of 100 to get read records and overlapping intervals will be combined. Interval files should be passed using the –interval-file option. This option can be used multiple times. e.g. -L chr1 -L chr2:10000 -L chr3:20000+ -L chr4:10000-20000 (default: None)

--interval-padding

(-ip) Padding size (in base pairs) to add to each interval you are including (default: None)

--out-recal-file

(required) Output BQSR Report File.

--num-gpus

Defaults to number of GPUs in the system. Number of GPUs to use for a run.

--gpu-devices

Which GPU devices to use for a run. By default, all GPU devices will be used. To set specific GPU devices, enter a comma-separated list of GPU device numbers.

applybqsr updates the Base Quality Scores using the BQSR report.

QUICK START-CLI

            

            
$ pbrun applybqsr --ref Ref/Homo_sapiens_assembly38.fasta \
--in-bam mark_dups_gpu.bam \
--in-recal-file recal_gpu.txt  \
--out-bam S1_updated.bam \
        

COMPATIBLE GATK4 COMMAND

The command below is the GATK4 counterpart of the Parabricks command above. The output from these commands will generate the exact same results as the output from the above command.

            

            
$ gatk ApplyBQSR --java-options -Xmx30g -R Ref/Homo_sapiens_assembly38.fasta \
-I=mark_dups_gpu.bam --bqsr-recal-file=recal_cpu.txt  -O=S1_updated.bam
        

OPTIONS

--ref

(required) The reference genome in fasta format. We assume that the indexing required to run bwa has been completed by the user.

--in-bam

(required) Path to the input BAM file.

--in-recal-file

(required) Input BQSR Report File.

--out-bam

(required) Output BAM file.

--interval-file

Path to an interval file for BQSR step with possible formats: Picard-style (.interval_list or .picard), GATK-style (.list or .intervals), or BED file (.bed). This option can be used multiple times (default: None)

--interval

(-L) All intervals will have a padding of 100 to get read records and overlapping intervals will be combined. Interval files should be passed using the –interval-file option. This option can be used multiple times. e.g. -L chr1 -L chr2:10000 -L chr3:20000+ -L chr4:10000-20000 (default: None)

--interval-padding

(-ip) Padding size (in base pairs) to add to each interval you are including (default: None)

--num-threads

Defaults to 8. Number of threads for worker.

--num-gpus

Defaults to number of GPUs in the system.

Number of GPUs to use for a run.

--gpu-devices

Which GPU devices to use for a run. By default, all GPU devices will be used. To set specific GPU devices, enter a comma-separated list of GPU device numbers.