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Note:The below workflow is for sequence level analysis for RNAseq data (16SrRNA).

Objectives

Here, we show how to (1) assign taxonomy (2) compare phylogenetic relationship among sequences (3) predict genes and functions. This SOP focuses on overcoming memorycap issues with USEARCH 32-bit version. The included scripts can be used to perform these objectives.

Equipment:

Linux or Unix 64-bit OS, > 20GB RAM and > 4 CPU cores.

Script-flow:

convert_fq_fa.sh -> merge-paired-reads.sh -> creat_OTU.sh -> create_phylo.sh -> pred_genes.sh

Tools/requirements:

  1. USEARCH 7.0.1001: http://www.drive5.com/usearch/download.html and usearch version 6.1.554 # rename USEARCH61 Make sure usearch version 6.1.554 is installed in your path (moved to /usr/bin) or you run it your dir sudo chmod 777 /usr/bin/usearch61 #make executable . Both USEARCH are required

  2. QIIME 64-bit of the latest qiime should be installed along with its dependencies. Cent OS installation guide: http://www.chenlianfu.com/?p=2362 and https://gist.github.com/alanorth/100dd95f809223ae0100

  3. download reference database http://drive5.com/uchime/gold.fa

  4. download UPARSE scripts: http://drive5.com/python/python_scripts.tar.gz

  5. download Faster_splitter: http://kirill-kryukov.com/study/tools/fasta-splitter/

  6. download Greengenes Database ftp://greengenes.microbio.me/greengenes_release/gg_13_5/gg_13_8_otus.tar.gz