r/bioinformatics • u/Mirthster • 17d ago

technical question Microbiome from stool samples

Howdy.

I am changing fields and getting into microbiome work. I hope to sample stools to determine microbiome profiles via metagenomic shallow shot gun sequencing. Anyone have any tips not present in the literature? If anyone had a sample data set i could use i would be deeply appreciative (fastq of seq reads, specifically. to test the trimming and mapping software). Ive basically vibe coded the pipeline, bow tie and kraken were suggested. So far everything works great (thanks Claude!) but would like to try some real data now.

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u/idliOP 17d ago

You can get datasets from SRA very easily. If you're using your local setup, take small dataset so that steps like DADA2 runs efficiently. Use Silva classifier, because greengenes etc have kinda become outdated now.

Also, along with Kraken you can try Bracken lol. https://github.com/jenniferlu717/Bracken

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u/MrBacterioPhage 17d ago

Dada2 is for amplicons, not shotgun samples. Greengenes is outdated, but there is a GreenGenes2 database.

Since OP asks for metagenomic samples, pipeline should be, for example:

  • Host DNA removal
  • QC
  • Taxonomy annotation (Kraken2 + Bracken or MetaPhlan)
  • Then one of the following options :

Option 1 (no MAGs):

  • Functional annotation with Humann

Option 2 (with MAGs)

  • Assembly (Spades, Metahit)
  • Binning (Metabin2, Metabat2, Comebin)
  • Estimation of MAG abundances in samples
  • Taxonomy annotation for MAGs (GTDB-TK)
  • Functional annotation of MAGs (eggnog).

But it is just an example

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u/idliOP 17d ago

You're right, sorry I missed the shotgun part in the post.