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Nicotine-Microbiome

IQ Biology Rotation Project with Noah Fierer

Goal

  • Assess number of nicotine-degrading genes in the oral microbiomes of smokers/vapers vs non-smokers/vapers

Methods

  • Pull shotgun metagenomic data from this paper
    • Project IDs: PRJNA548383, PRJNA544061, and PRJNA508385
  • Create database of sequences for genes involved in nicotine degradation, based on Mu et al., 2020
    • MetaCyc degradation pathways tsvs added
    • Pull fastas from GenBank
      • conda install -y -c conda-forge -c bioconda -c defaults entrez-direct
      • esearch -db protein -query 'Protein name' | efetch -format fasta
      • But some of the genes only have names, not accession IDs, so they return sequences for unwanted genes...
    • Start with a few key genes to get the pipeline running
    • Pull from UniProt and/or GenBank
    • Then add others once things are working
    • Check for these genes in their hosts in IMG as a sanity check
  • Use Diamond to find nicotine-degrading genes in the oral microbiolmes
  • Assess difference in nicotine-degrading genes in the oral microbiomes of smokes/vapers vs controls
  • Look at MAGs if time?

Database

  • Get MetaCyc gene details
  • Search E.C. IDs in UniProt
  • Download fastas for all matching sequences
    • For 3.5.1.3, filtered only bacteria because lots of human and mouse sequences were coming up
    • Skipped 1.3.99.- and 1.5.99.- because too many unrelated genes were coming up
    • E.C. for nctB was sourced from Uniprot info on (S)-6-hydroxynicotine oxidase
    • Skipped 1.5.8.M1 because it wasn't pulling up any results, and searching the name pulled up kdhA/B/C, which I already had

Diamond

  • Please cite http://dx.doi.org/10.1038/s41592-021-01101-x Nature Methods (2021)
  • tested diamond with diamond blastx -q PRJNA548383/SRR9641933/SRR9641933_1.fastq -d database-building/uniprot-nicotine-db.dmnd -o test.tsv
    • Got a couple hundred hits

Files/details

  1. setup_scripts contains scripts with code to install sra toolkit. I haven't tried these in script format (since I only needed to install it once), but I imagine I will need this code later if using fastq-dump on fiji.
  • If having issues see the documentation or config guidelines.
  • Note for future John can try the following instead of vdb-config -i:
    • vdb-config --restore-defaults
    • vdb-config -s "/repository/user/main/public/root=."
    • vdb-config -s "cache-enabled=true"
    • vdb-config --report-cloud-identity yes
  1. get_data.sh is a script to loop through the SRA accession values for the projects and download the fastq files in directories named by their run IDs.
  • If having issues with this, consider something similar to the following:

while read line; do
wget http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?cmd=dload&run_list=${line}&format=fastq;
done<list_of_ids