Scripts to facilitate processing of 3RAD data with STACKs.

Demultiplexing

1. Demultiplex iTru7 indexes

Prepare a tab delimited barcodes file with 8 bp index sequence in the first column and ID in the second column. Run the process-radtags-i7.sh script.

process-radtags-i7.sh \
  <path to read 1 sequences> \
  <path to read 2 sequences> \
  <path to barcodes file> \
  <desired output directory path> # Cannot already exist

2. Declone

Run clone_filter.py script.

clone_filter.py \
  <path to process_radtags output director> \
  <desired output directory path> # Cannot already exist

3. Concatenate

Concatenate output from the clone_filter.py script sharing 3RAD adapter indexes (i.e. contains samples from the same project).

Example:

cat <i7-index1_R1.fq.gz> <i7_index2_R2.fq.gz> ... project1_R1.fq.gz
cat <i7-index1_R1.fq.gz> <i7_index2_R2.fq.gz> ... project1_R2.fq.gz

4. Demultiplex samples with 3RAD adapter indexes

Prepare a barcodes file with the index sequences of the 3RAD adapters and the corresponding sample IDs. A script to facilitate the generation of a barcodes file can be found at https://github.com/kerrycobb/radseq-barcodes-file-gen

Run process-radtags-samples.sh

process-radtags-samples.sh \
  <path to concatenated read 1 reads> \
  <path to concatenated read 2 reads> \
  <path to barcodes file> \
  <desired output directory path> \ # Cannot already exist
  <enzyme 1> \
  <enzyme 2>

Stacks Alignment

1. Run ustacks

You will need to modify this script to work with your own HPC system.

run-ustacks.fish \
  <demultiplexed sample directory> \
  <output directory> \
  <populations file> \
  <value for ustacks M param>

2. Run cstacks, sstacks, tsv2bam, and gstacks

run-stacks-pipe.fish \
  <ustacks directory> \
  <populations file> \
  <value for cstacks n param>

kerrycobb/rad-pipeline