This pipeline is meant to provide an easy interface and automation for running Drop-seq tools, Picard, and some new scripts developed here. Drop-seq tools is developed by James Nemesh and the McCarroll lab at the Harvard Medical School (http://mccarrolllab.com/dropseq). The procedure is outlined here: http://mccarrolllab.com/wp-content/uploads/2016/03/Drop-seqAlignmentCookbookv1.2Jan2016.pdf
| File | Description |
|---|---|
| dropseq | main program |
| config.txt | global parameters and file paths |
| get_bam_trans_reads_counts.pl | extracts gene and read counts from aligned BAM |
| plot_trans_reads_counts.r | generate plots of cumulative gene/read distributions |
Drop-seq pipeline v0.1 - Creaded by John P. McCrow (4/12/2018)
Usage: dropseq [command] (options)
Commands:
fastq2bam convert paired FASTQ to sorted BAM
tagXC add cell barcodes to BAM, XC tag
tagXM add molecular barcodes to BAM, XM tag
filter filter out low quality barcode reads by XQ tag
trimAdapter trim partial SMART adapter from 5'-end
trimPolyA trim partial poly-A from 3'-end
bam2fastq convert trimmed/filtered BAM to FASTQ for use in alignment
starAlign alignment to reference using STAR
sortSam sort aligned SAM and convert to BAM
merge merge unaligned and aligned BAM files
tagGenes tag exons/genes in BAM, GE tag
correctXC correct bead synthesis errors
histogram plot cumulative read and gene distributions
dge digital gene expression per cell barcode
cleanup remove some intermediate files
make create make file to automate entire pipeline
Options:
--help, -h show this help page
--list, -l file file with list of sample names
runs sample number set in SGE_TASK_ID
--sample, -s name library/sample name (base filename)
one of -l or -s is required
--rm remove output file from previous step when finished
* See command help for command specific options
- Make sure all dependencies are installed (see below).
- Edit the config.txt file to set file paths. It is also possible to add a separate copy of config.txt to your current directory when running dropseq which will override the global config.txt.
- Test dropseq to see if all dependancies are found
dropseq test
- Perl (https://www.perl.org/get.html)
- Java 1.8+ (http://www.oracle.com/technetwork/java/javase/downloads/index.html)
- Drop-seq Tools (http://mccarrolllab.com/download/1276)
- Picard 2+ (http://broadinstitute.github.io/picard)
- STAR Aligner (https://github.com/alexdobin/STAR/releases)