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

Docs: http://migec.readthedocs.org/en/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/

Docs: http://mitcr.milaboratory.com/documentation/

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