Pipeline example: MiSeq 2x300 fungal ITS
OTU / denoising
Example pipelines with test
This tutorial uses data from study
deposited by CEH at the European
Nucleotide Archive derived from samples of Scots Pine (Pinus sylvestris)
needles collected from forests and plantations in Scotland. Libraries were
constructed using fITS7 (forward) and ITS4 (reverse) primers described in
al. (2012) targeting the 58S and LSU rRNA genes flanking the ITS2
region. Sequencing was done using MiSeq 2x300 PE. To reduce the dataset size
for this tutorial, 5,000 reads were taken at random from the first ten pairs
of FASTQ files.
You need to set
the environment variable $usearch to the name (or path) for your
usearch binary file. The bash script
assumes that the FASTQ files and the sintax reference database are stored in
a directory called ../data/. Output is written to a directory called ../out/
which is deleted and re-built each time the script is run. I suggest making a project directory, say
ex_miseq_its, with sub-directories named data/ and scripts/. Put the ex_miseq.bash
script in the scripts/ sub-directory and execute it from there.
Download commands and data
Reads: right-click on
and click Save As. Use tar -zxvf *.tar.gz to extract.
reference database: right-click on
rdp_16s_v16.fa.gz and click Save As. Use gunzip rdp_16s_v16.fa.gz
Bash script: select all text below (ctrl+A) and
copy/paste into a text editor, or right-click on ex_miseq_its.bash and click Save As.