Prerequisites¶
Datasets¶
The tutorial contains the following datasets:
- Raw sequencing reads (paired-end FASTQ) coming from
two samples taken from the same individual,
1000 T-cells each. Together with them comes
barcodes.txt
storing information for de-multiplexing. To be used in Part 1. - Diversity estimates generated by VDJtools for data from 39 donors of various age. To be used in Part 2 and 3
Datasets are stored in the tutorial repository, to download them run:
git clone https://github.com/mikessh/repseq-tutorial
they can be found in part1/
and part2/
folders respectively.
Software¶
All software tools necessary for this tutorial are stored in the
repseq-tutorial
repository as well.
Warning
Java v1.8+ is required to run these tools.
MIGEC¶
A pipeline for processing of RepSeq data, focused on UMI-tagged reads. There are 5 steps of MIGEC pipeline:
- Checkout De-multiplexing, UMI sequence extraction, read re-orientation, adapter trimming
- Histogram UMI coverage statistics
- Assemble Assemble consensus sequences from reads tagged with the same UMI. Filters out low-coverage and erroneous UMIs.
- CdrBlast V-(D)-J mapping and clonotype assembly
- FilterCdrBlastResults Additional correction of hot-spot errors.
Latest binaries: https://github.com/mikessh/migec/releases/latest
MITCR¶
A software for fast and robust processing of TCR sequencing data. The software performs V-(D)-J mapping, clonotype assembly with frequency-based error correction and low-quality read rescue via re-mapping to assembled clonotypes.
Latest binaries: http://mitcr.milaboratory.com/downloads/
VDJtools¶
A framework for post-analysis of immune repertoire sequencing data. Includes 20+ analysis modules: from simple routines such as spectratyping to diversity estimation and repertoire clustering modules.
Latest binaries: https://github.com/mikessh/vdjtools/releases/latest
Docs: http://vdjtools-doc.readthedocs.org/en/latest/modules.html
Important
As VDJtools performs some plotting via R, so it can throw an
error that a specific R package is lacking. Those packages
should be installed with install.packages
command
in R shell.
Alternatively, one can run java -jar vdjtools-1.0.2.jar RInstall
to allow VDJtools intall all R packages it uses (will take a substantial amount of time).
The list of required packages:
reshape, ggplot2, gridExtra, FField,
ape, reshape2, MASS, plotrix, RColorBrewer,
scales