Dr.seq is a quality control (QC) and analysis pipeline for Drop-seq data. It takes a fastq file with barcode data and a fastq file of reads along with supporting files (annotation and indices for alignment) to provide QC data at the level of reads, individual cells, bulk cells, and cell-clustering.
There are changes to the command line interface between versions. Documentation here should refer to the newest version.
There may be multiple versions of Dr.seq available. An easy way of selecting the version is to use modules. To see the modules available, type
module avail drseq
To select a module use
module load drseq/[version]
where [version] is the version of choice.
drseq is a multithreaded application. Make sure to match the number of cpus requested with the number of threads.
Environment variables set
- $PATH
Dependencies
- bowtie2 or STAR
- macs 1.4 (for Drop-ChIP and ATAC-Seq data)
- R
- bedtools
- samtools
Drseq can use either bowtie2 or STAR as a short read mapper. Since the choice is up to the user, neither of these modules is loaded by default. Please load the correct module for your analysis manually.
When analyzind Drop-ChIP or ATAC-Seq data please load the mac 1.4 module.
R, samtools and betools are loaded automatically.
References
- X. Huo, S. Hu, C. Zhao, Y. Zhang. Dr.seq: a quality control and analysis pipeline for droplet sequencing.. Bioinformatics 2016, 32:2221-2223. Pubmed | PMC | Journal
Documentation
Allocate an interactive session and run the program. Sample session:
[user@biowulf]$ sinteractive --cpus-per-task=6 --mem=10g 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 bowtie drseq [user@cn3144 ~]$ DrSeq simple \ -b drseq_test_1.fastq \ -r drseq_test_2.fastq \ -n test -f \ -g $PWD/mm10_refgenes.txt \ --maptool bowtie2 \ --mapindex /fdb/igenomes/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome \ --cellbarcoderange 1:12 \ --umirange 13:20 \ --clean \ --thread $SLURM_CPUS_PER_TASK Start Drseq Step0: Data integrate Detected input file format is fastq use bowtie2 as alignment tools option setting: mapping thread is 4 Step0 Data integrate DONE Step1: alignment [...snip...] [user@cn3144 ~]$ exit salloc.exe: Relinquishing job allocation 46116226 biowulf$
Create a batch script similar to the following example:
#! /bin/bash # this file is drseq_batch.sh module load bowtie module load drseq Drseq.py simple \ -b drseq_test_1.fastq \ -r drseq_test_2.fastq \ -n test_out -f \ -g $PWD/mm10_refgenes.txt \ --maptool bowtie2 \ --mapindex /fdb/igenomes/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome \ --cellbarcoderange 1:12 \ --umirange 13:20 \ --clean \ --thread $SLURM_CPUS_PER_TASK
Note that in this example used data obtained from the Dr.seq home page as well as annotation obtained from the UCSC browser.
Submit to the queue with sbatch:
biowulf$ sbatch --cpus-per-task=6 --mem=10g drseq_batch.sh
Create a swarm command file similar to the following example:
# this file is drseq.swarm DrSeq simple \ -b sample1_1.fastq \ -r sample1_2.fastq \ -n sample1 -f \ -g $PWD/mm10_refgenes.txt \ --maptool bowtie2 \ --mapindex /fdb/igenomes/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome \ --cellbarcoderange 1:12 \ --umirange 13:20 \ --clean \ --thread $SLURM_CPUS_PER_TASK DrSeq simple \ -b sample2_1.fastq \ -r sample2_2.fastq \ -n sample2 -f \ -g $PWD/mm10_refgenes.txt \ --maptool bowtie2 \ --mapindex /fdb/igenomes/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome \ --cellbarcoderange 1:12 \ --umirange 13:20 \ --clean \ --thread $SLURM_CPUS_PER_TASK
And submit to the queue with swarm
biowulf$ swarm -f drseq.swarm -g10 -t6 --module drseq --module bowtie