Long Ranger2.1, printed on 10/13/2024
Analysis software for 10x Genomics linked read products is no longer supported. Raw data processing pipelines and visualization tools are available for download and can be used for analyzing legacy data from 10x Genomics kits in accordance with our end user licensing agreement without support. |
Long Ranger can be used to analyze multiple libraries. Depending on your exact scenario, the approach may be different. Here are two different circumstances:
You created one library, but sequenced it more than once. This might have been to increase coverage depth, or for some other reason. You might have sequenced the library across different lanes of the same flowcell, or across multiple flow cells. In any of these cases, provided that this all comes from a single library, you can analyze these runs as a single sample using longranger wgs or targeted by Specifying Input FASTQs.
You created multiple libraries from the same sample. If it is necessary to pool multiple libraries and analyze them as a single sample, you can do that. It just requires learning a little more about exactly how Long Ranger works, and writing a customized pipeline configuration file, called an MRO. This page covers this final scenario.
Long Ranger uses a pipeline management framework called Martian. The pipeline and each stage in it is specified by a configuration file called an MRO file. Usually, the longranger commands create the appropriate MRO files for you, but in the case that you want to do something outside the normal workflows, it is possible to create a custom MRO file to directly exercise the full range of features.
In the example below we describe how to construct an MRO file to specify multiple libraries as well as multiple flow cells (since most often the multiple libraries will have been sequenced on different runs).
The easiest way to write your own MRO is to start with the MRO from a previous pipeline. Assuming you have already run an analysis on the same pipeline (meaning, wgs
or targeted
as appropriate) on a single-flowcell sample (e.g., sample345), examine the _invocation file contained in its output directory.
Note: this example assumes that the input flowcells were processed with longranger mkfastq.
$ cat sample345/_invocation @include "phaser_svcaller_cs.mro" call PHASER_SVCALLER_CS( fastq_mode = "BCL_PROCESSOR", sample_id = "sample345", sample_def = [ { "bc_in_read": 1, "bc_length": 16, "gem_group": null, "lanes": null, "read_path": "/home/jdoe/runs/HBA2TADXX", "sample_indices": [ "any" ] } ], reference_path = "/opt/refdata-hg19-2.1.0", sample_desc = "", sex = "f", targets = null, vc_mode = "freebayes", vc_ground_truth = null, restrict_locus = null )
The sample_def argument controls the parameters used to define this sample and is a JSON-encoded list of maps that define:
null
.Make a copy of this _invocation file; this will be the MRO from which we will build our multi-library invocation MRO.
Continuing with the example MRO above, we would make the following changes:
$ cp sample345/_invocation sample345-multi.mro $ nano sample345-multi.mro ... $ cat sample345-multi.mro @include "phaser_svcaller_cs.mro" call PHASER_SVCALLER_CS( fastq_mode = "BCL_PROCESSOR", sample_id = "sample345-multi", sample_def = [ { "bc_in_read": 1, "bc_length": 16, "gem_group": 1, "lanes": null, "read_path": "/home/jdoe/runs/HAWT7ADXX", "sample_indices": [ "any" ] }, { "bc_in_read": 1, "bc_length": 16, "gem_group": 2, "lanes": null, "read_path": "/home/jdoe/runs/HAWPUADXX", "sample_indices": [ "any" ] } ], sample_desc = "", reference_path = "/opt/refdata-hg19-2.1.0", sex = "f", targets = null, vc_mode = "freebayes", vc_ground_truth = null, restrict_locus = null )
The cellular barcode sequences will include suffixes from the different gem groups, i.e. libraries.
AGAATGGTCTGCAT-1 CTGATCGATATCGA-1 GTAGCAACGTCGTA-2 AGAATGGTCTGCAT-2
This is how Long Ranger prevents the same barcode from different sets of molecules in different libraries from being erroneously combined based only on the barcode sequence.
Once you have this single-sample, multi-library, multi-flowcell MRO, confirm that its syntax is valid with longranger mrc, the MRO compiler included with Long Ranger:
$ longranger mrc sample345-multi.mro Successfully compiled 1 mro files.
A MRO ParseError: unexpected token error suggests that your sample_def entry is not valid JSON. Ensure that your commas are present if required (and absent if required) after each item. |
Then run the MRO file using longranger's alternate MRO-mode syntax:
$ longranger wgs sample345-multi sample345-multi.mro --uiport=3600 Martian Runtime - 2.2.2 Serving UI at http://localhost:3600 Running preflight checks (please wait)... 2016-05-01 12:00:00 [runtime] (ready) ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._ALIGNER.SETUP_CHUNKS 2016-05-01 12:00:00 [runtime] (run:local) ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH 2016-05-01 12:00:00 [runtime] (run:local) ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH.fork0.chnk0.main
where