Computational protocol: Archaea and Fungi of the Human Gut Microbiome: Correlations with Diet and Bacterial Residents

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Protocol publication

[…] Sequences obtained were decoded and quality controlled using the QIIME pipeline . OUT’s were formed at 97% and 95% similarity for archaeal and fungal sequences respectively, and were considered for further analysis if they had a minimum of 5 sequences detected across all samples. Taxonomy was assigned to OTU representative sequences using the RDPclassifier for archaeal sequences and BROCC for fungal sequences. All taxonomy assignments were manually curated to check for accuracy and nomenclature using BLASTn against GenBank’s NR/NT database. For Archaea, the taxonomic assignment given by RDP to 2 out of the 12 archaeal OUT’s detected was corrected due to low coverage of those groups on RDP. For ITS, of the 290 OUT’s detected, 10 were missing mid-level (between phylum and genus) taxonomic information, which was filled in, and 5 yielded differing mid level taxonomies, of which only one of per genus was used. Two ITS OUT’s were automatically classified down to genus using BROCC, but had their taxonomy assignment brought up to Order level upon inspection of BLAST results (both OUT’s were present in one sample each). OTU sequence counts for each sample were aggregated at genus level. All downstream analysis was done at the Genus level using R unless otherwise noted. Genera were considered in the analysis if present in at least 9 out of the 96 available samples, and its absolute sequence count was equal to or greater than 10. For the ITS amplicon, samples were included in downstream analysis if they yielded at least 200 sequences. All novel sequence data was deposited at NCBI’s Sequence Read Archive under accession number SRP021021. […]

Pipeline specifications

Software tools QIIME, BROCC, BLASTN
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Chemicals Carbohydrates, Fatty Acids