10x Genomics
Chromium Genome & Exome

Long Ranger2.1, printed on 11/22/2024

Whole Genome Phasing and SV Calling

Long Ranger's Whole Genome Mode analyzes sequencing data from a Chromium-prepared library. This involves the following steps:

1. Run longranger mkfastq on the Illumina BCL output folder to generate FASTQ files.

2. Run longranger wgs for each sample that was demultiplexed by longranger mkfastq.

For the following example, assume that the Illumina BCL output is in a folder named /sequencing/140101_D00123_0111_AHAWT7ADXX.

Run longranger mkfastq

First, follow the instructions on running longranger mkfastq to generate FASTQ files. For example, if the flowcell serial number was HAWT7ADXX, then longranger mkfastq will output FASTQ files in HAWT7ADXX/outs/fastq_path.

Run longranger wgs

To run Long Ranger in whole genome mode, you use the longranger wgs command, with the following common parameters. For a complete list of command-line options, run longranger wgs --help.

After determining these input arguments, call longranger wgs:

$ cd /home/jdoe/runs
$ longranger wgs --id=sample345 \
                 --reference=/opt/refdata-hg19-2.1.0 \
                 --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path

Following a set of preflight checks to validate input arguments, Long Ranger pipeline stages will begin to run:

longranger wgs 2.1.6
Copyright (c) 2016 10x Genomics, Inc.  All rights reserved.
-----------------------------------------------------------------------------
Martian Runtime - 2.2.2
 
Running preflight checks (please wait)...
2016-05-01 12:00:00 [runtime] (ready)           ID.sample345.PHASER_SVCALLER_CS.PHASER_SVCALLER._ALIGNER.SETUP_CHUNKS
2016-05-01 12:00:00 [runtime] (run:local)       ID.sample345.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH
2016-05-01 12:00:00 [runtime] (run:local)       ID.sample345.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH.fork0.chnk0.main
...

By default, longranger wgs will use all of the cores available on your system to execute pipeline stages. You can specify a different number of cores to use with the --localcores option; for example, --localcores=16 will limit the pipeline to using up to sixteen cores at once. Similarly, --localmem will restrict the amount of memory (in GB) used by longranger wgs.

The pipeline will create a new folder named with the sample ID you specified (e.g. /home/jdoe/runs/sample345) for its output. If this folder already exists, Long Ranger will assume it is an existing pipestance and attempt to resume running it.

Variant Calling

By default, longranger wgs calls SNPs and indels with Freebayes, phases those variants, and adds structural variant calls. GATK may also be used.

Alternatively, you may provide an existing VCF file as an input to the pipeline. In this "pre-called VCF" mode, longranger wgs does not call SNPs and indels itself, but phases the variants in the supplied VCF file, and also outputs structural variant calls.

As of Long Ranger 2.0, longranger wgs no longer supports setting the --vcmode argument when supplying a pre-called VCF file.

Output Files

A successful longranger wgs execution should conclude with a message similar to this:

2016-05-02 15:46:41 [runtime] (run:local)       ID.sample345.PHASER_SVCALLER_CS.PHASER_SVCALLER.LOUPE_PREPROCESS.fork0.join
2016-05-02 15:46:44 [runtime] (join_complete)   ID.sample345.PHASER_SVCALLER_CS.PHASER_SVCALLER.LOUPE_PREPROCESS
2016-05-02 15:46:55 [runtime] VDR killed 4738 files, 223GB.
 
Outputs:
- Run summary:               /home/jdoe/runs/sample345/outs/summary.csv
- BAM barcoded:              /home/jdoe/runs/sample345/outs/phased_possorted_bam.bam
- BAM index:                 /home/jdoe/runs/sample345/outs/phased_possorted_bam.bam.bai
- VCF phased:                /home/jdoe/runs/sample345/outs/phased_variants.vcf.gz
- VCF index:                 /home/jdoe/runs/sample345/outs/phased_variants.vcf.gz.tbi
- Large-scale SV calls:      /home/jdoe/runs/sample345/outs/large_sv_calls.bedpe
- Large-scale SV candidates: /home/jdoe/runs/sample345/outs/large_sv_candidates.bedpe
- Large-scale SVs:           /home/jdoe/runs/sample345/outs/large_svs.vcf.gz
- Large-scale SVs index:     /home/jdoe/runs/sample345/outs/large_svs.vcf.gz.tbi
- Mid-scale deletions:       /home/jdoe/runs/sample345/outs/dels.vcf.gz
- Mid-scale deletions index: /home/jdoe/runs/sample345/outs/dels.vcf.gz.tbi
- Loupe file:                /home/jdoe/runs/sample345/outs/loupe.loupe
 
Pipestance completed successfully!

The output of the pipeline will be contained in a folder named with the sample ID you specified (e.g. sample345). The subfolder named outs will contain the main pipeline output files:

Once longranger wgs has successfully completed, you can browse the resulting .loupe file in the Loupe genome browser, or refer to the Understanding Output section to explore the data by hand.