Command-line Options

Perform the following command-line options using the dragen:

•

Build Reference/Hash Table

dragen --build-hash-table true --ht-reference <REF_FASTA> \
--output-directory <REF_DIRECTORY> [options]

•

Run Map/Align and Variant Caller (*.fastq to *.vcf)

dragen -r <REF_DIRECTORY> --output-directory <OUT_DIRECTORY> \
--output-file-prefix <FILE_PREFIX> [options] -1 <FASTQ1> \
[-2 <FASTQ2>] –-RGID <RG0> --RGSM <SM0> --enable-variant-caller true

•

Run Map/Align (*.fastq to *.bam)

dragen -r <REF_DIRECTORY> --output-directory <OUT_DIRECTORY> \
--output-file-prefix <FILE_PREFIX> [options] \
-1 <FASTQ1> [-2 <FASTQ2>] \
--RGID <RG0> --RGSM

•

Run Variant Caller (*.bam to *.vcf)

dragen -r <REF_DIRECTORY> --output-directory <OUT_DIRECTORY> \
--output-file-prefix <FILE_PREFIX> [options] -b <BAM> \
--enable-variant-caller true

•

Run BCL Converter (BCL to *.fastq)

dragen --bcl-conversion-only true --bcl-input-directory <BCL_DIRECTORY> \
--output-directory <OUT_DIRECTORY>

•

Run RNA Map/Align (*.fastq to *.bam)

dragen -r <REF_DIRECTORY> --output-directory <OUT_DIRECTORY> \
--output-file-prefix <FILE_PREFIX> [options] -1 <FASTQ1> \
[-2 <FASTQ2>] --enable-rna true

DRAGEN has two primary modes of operation, as follows:

•

Mapper/aligner

•

Variant caller

DRAGEN is capable of performing each mode independently or as an end-to-end solution. DRAGEN also allows you to enable and disable decompression, sorting, duplicate marking, and compression along the DRAGENpipeline.

Before you can use the DRAGENsystem for aligning reads, you must load a reference genome and its associated hash tables onto the PCIe card. For information on preprocessing a reference genome’s FASTA files into the native DRAGEN binary reference and hash table formats, see Prepare a Reference Genome. You must also specify the directory containing the preprocessed binary reference and hash tables with the -r [or --ref-dir] option. This argument is always required.

Use the following command to You can load the reference genome and hash tables to DRAGEN card memory separately from processing reads/, as follows.

dragen -r /staging/human/reference/hg19/hg19.fa.k_21.f_16.m_149

You can Uuse the -l (--force-load-reference) option to force the reference genome to load even if it is already loaded, as follows.

dragen -l -r /staging/human/reference/hg19/hg19.fa.k_21.f_16.m_149

The time needed to load the reference genome depends on the size of the reference, but for typical recommended settings, it takes approximately 30–60 seconds.

The following commandline options for output are mandatory:

•

--output-directory <out_dir>—Specifies the output directory for generated files.

•

--output-file-prefix <out_prefix>—Specifies the output file prefix. DRAGENappends the appropriate file extension onto this prefix for each generated file.

•

-r [ --ref-dir ] specifies the reference hash table.

For brevity, the following examples do not include these mandatory options.

For mapping and aligning, the output is sorted and compressed into BAM format by default before saving to disk. You can control the output format from the map/align stage with the --output-format <SAM|BAM|CRAM> option. If the output file exists, DRAGEN issues a warning and exits. To force overwrite if the output file already exists, use the -f [ --force ] option.

For example, the following commands output to a compressed BAM file and forces overwrite:

dragen ... -f

dragen ... -f --output-format bam

To generate a BAI-format BAM index file (.bai file extension), set --enable-bam-indexing to true.

The following example outputs to a SAM file and forces overwrite:

dragen ... -f --output-format sam

The following example outputs to a CRAM file and forces overwrite:

dragen ... -f --output-format cram

DRAGEN can generate mismatch difference (MD) tags, as described in the BAM standard. The feature is turned off by default because there is a small performance cost to generate these strings. To generate MD tags, set --generate-md-tags to true.

To generate ZS:Z alignment status tags, set --generate-zs-tags to true. These tags are only generated in the primary alignment and when a read has suboptimal alignments qualifying for secondary output (even if none were output because --Aligner.sec-aligns was set to 0). 

The following are valid tag values:

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ZS:Z:R—Multiple alignments with similar score were found.

•

ZS:Z:NM—No alignment was found.

•

ZS:Z:QL—An alignment was found, but it was below the quality threshold.

To generate SA:Z tags, set --generate-sa-tags to true (the default). These tags provide alignment information (position, cigar, orientation) of groups of supplementary alignments, which are useful in structural variant calling.

The Picard Base Quality Score Recalibration (BQSR) tool produces output BAM files that include tags BI and BD. BQSR calculates these tags relative to the exact sequence for a read. If a BAM file with BI and BD tags is used as input to mapper/aligner with hard clipping enabled, the BI and/or BD tags can become invalid.

The recommendation is to strip these tags when using BAM files as input. To remove the BI and BD tags, set the --preserve-bqsr-tags option to false. If you preserve the tags, DRAGEN warns you to disable hard clipping. 

DRAGEN assumes that all the reads in a given FASTQ belong to the same read group. The DRAGEN system creates a single @RG read group descriptor in the header of the output BAM file, with the ability to specify the following standard BAM attributes:

Attribute	Argument	Description
ID	--RGID	Read group identifier. If you include any of the read group parameters, RGID is required. It is the value written into each output BAM record.
LB	--RGLB	Library.
PL	--RGPL	Platform/technology used to produce the reads. The BAM standard allows for values CAPILLARY, LS454, ILLUMINA, SOLID, HELICOS, IONTORRENT and PACBIO.
PU	--RGPU	Platform unit, eg, flowcell-barcode.lane.
SM	--RGSM	Sample.
CN	--RGCN	Name of the sequencing center that produced the read.
DS	--RGDS	Description.
DT	--RGDT	Date the run was produced.
PI	--RGPI	Predicted mean insert size.

If any of these arguments are present, the DRAGEN software adds an RG tag to all the output records to indicate that they are members of a read group. The following example shows a command line that includes read group parameters:

When using the --fastq-list option to input multiple read groups, BAM tags (and others) are specified for each read group by adding columns to the fastq_list.csv file. Each column heading consists of four capital letters and each begins with 'RG'. For each column, each read group’s values for that column are propagated to the output BAM file in an identically named tag.

To suppress the license status message at the end of the run, use the --lic-no-print option. The following shows an example of the license status message:

LICENSE_MSG| =====================================================
LICENSE_MSG| License report
LICENSE_MSG|   Genome status [ACxxxxxxxxxxx] : used 1263.9 Gbases since 2018-Feb-15 (1263886160894 bases, unlimited)
LICENSE_MSG|   Genome  bases [ACxxxxxxxxxxx] : 202000000
LICENSE_MSG|   Genome  bases [total]         : 202000000