Wiki¶

Data location¶

This project was run on the Ilifu server

/cbio/users/katie/Hemmings

Testing data raw reads¶

/cbio/users/katie/Hemmings/fastqs

EMIRGE software setup¶

[[[https://github.com/csmiller/EMIRGE]]] software was setup as a singularity container, using a Docker image available on Dockerhub [[[https://hub.docker.com/r/golob/emirge]]].

1. From CBIO's BST server (katie@bst.cbio.uct.ac.za) I pulled the docker container with docker pull golob/emirge
2. The singularity container was built with rsync -avvP -e "ssh -i /home/katie/.ssh/id_rsa" singularity-containers/emirge_latest.simg katiel@transfer.ilifu.ac.za:/cbio/users/katie/Hemmings/containers
3. The singularity container was transferred to Ilifu with rsync -avvP -e "ssh -i /home/katie/.ssh/id_rsa" singularity-containers/emirge_latest.simg katiel@transfer.ilifu.ac.za:/cbio/users/katie/Hemmings/containers

EMIRGE troubleshooting¶

Unfortunately there were several hurdles setting up the SSU database for use with EMIRGE. Firstly, the emirge_makedb.py in the singularity container did not work. I had to do a git clone on the original github repo and edit the FTP site specified in the script from SILVA_{rel}SSURef_Nr99_tax_silva_trunc.fasta.gz to SILVA{rel}_SSURef_NR99_tax_silva_trunc.fasta.gz (Nr99 to NR99). This script is available at /cbio/users/katie/Hemmings/emirge_makedb.py
./emirge_makedb.py -p8 --silva-license-accepted. The steps executed in emirge_makedb.py are:

1) download the most recent SILVA SSU database, 2) filter it by sequence
length, 3) cluster at 97% sequence identity, 4) replace ambiguous bases
with random characters and 5) create a bowtie index.

Still, the above command aborted after the clustering step (step 3).I therefore had to manually replace ambiguous characters in the resulting clustered DB named SILVA_138.1_SSURef_NR99_tax_silva_trunc.ge1200bp.le2000bp.0.97.fasta, using the script from the github site (this script was not available in the singularity container, but can be found under /cbio/users/katie/Hemmings/EMIRGE/utils/fix_nonstandard_chars.py. The executed command was python2 EMIRGE/utils/fix_nonstandard_chars.py < SILVA_138.1_SSURef_NR99_tax_silva_trunc.ge1200bp.le2000bp.0.97.fasta > SILVA_138.1_SSURef_NR99_tax_silva_trunc.ge1200bp.le2000bp.0.97.fixed.fasta. Next, a bowtie index was built for this fasta file with . Note that these commands were still executed from within the Singularity container to make use of the necessary software installed therein.