Umitools on HPC

Umi-tools are tools for dealing with Unique Molecular Identifiers (UMIs)/Random Molecular Tags (RMTs) and single cell RNA-Seq cell barcodes. Currently there are 6 commands.

The extract and whitelist commands are used to prepare a fastq containg UMIs +/- cell barcodes for alignment.

Builds a whitelist of the 'real' cell barcodes
This is useful for droplet-based single cell RNA-Seq where the identity of the true cell barcodes is unknown. Whitelist can then be used to filter with extract (see below)

Flexible removal of UMI sequences from fastq reads.
UMIs are removed and appended to the read name. Any other barcode, for example a library barcode, is left on the read. Can also filter reads by quality or against a whitelist (see above)
The remaining commands, group, dedup and count/count_tab, are used to identify PCR duplicates using the UMIs and perform different levels of analysis depending on the needs of the user. A number of different UMI deduplication schemes are enabled - The recommended method is directional.

Groups PCR duplicates using the same methods available through `dedup`.
This is useful when you want to manually interrogate the PCR duplicates

Groups PCR duplicates and deduplicates reads to yield one read per group
Use this when you want to remove the PCR duplicates prior to any downstream analysis

Groups and deduplicates PCR duplicates and counts the unique molecules per gene
Use this when you want to obtain a matrix with unique molecules per gene. Can also perform per-cell counting for scRNA-Seq.

As per count except input is a flatfile


Important Notes

Interactive job
Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144 ~]$ module load umitools
[user@cn3144 ~]$ cp /usr/local/apps/umitools/example.fastq.gz . 
[user@cn3144 ~]$ umi_tools extract --stdin=example.fastq.gz --bc-pattern=NNNNNNNNN --log=processed.log --stdout processed.fastq.gz

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Batch job
Most jobs should be run as batch jobs.

Create a batch input file (e.g. For example:

set -e
module load umitools
umi_tools extract --stdin=example.fastq.gz --bc-pattern=NNNNNNNNN --log=processed.log --stdout processed.fastq.gz

Submit this job using the Slurm sbatch command.

sbatch [--mem=#]
Swarm of Jobs
A swarm of jobs is an easy way to submit a set of independent commands requiring identical resources.

Create a swarmfile (e.g. job.swarm). For example:

cd dir1; umi_tools extract --stdin=example.fastq.gz --bc-pattern=NNNNNNNNN --log=processed.log --stdout processed.fastq.gz
cd dir2; umi_tools extract --stdin=example.fastq.gz --bc-pattern=NNNNNNNNN --log=processed.log --stdout processed.fastq.gz
cd dir3; umi_tools extract --stdin=example.fastq.gz --bc-pattern=NNNNNNNNN --log=processed.log --stdout processed.fastq.gz

Submit this job using the swarm command.

swarm -f job.swarm [-g #] --module umitools
-g # Number of Gigabytes of memory required for each process (1 line in the swarm command file)
--module Loads the module for each subjob in the swarm