Illumina sequencing of human faecal sample
The V4 region of the bacterial 16S rRNA gene was amplified from each sample at external facilities (www.starseq.com). Briefly, triplicate end-point PCR reactions for the V4 region were performed using previously described primers (V4, 515fr-806rB [Klinworh et al 2013, Appril et al 2015, Walters et al 2015, Parada et al 2016]) and equimolarly pooled to reduce bias. Pooled libraries were gel purified and spiked with 15% PhiX prior to sequencing. Paired-end (2 x 300 base pair) high-throughput DNA sequencing was carried out using the MiSeq platform (Illuimna). Obtained reads were processed with the DADA2 pipeline and phyloseq R package (Callahan et al 2016, McMurdie et al 2013). Subsequently, a sequence table was constructed, taxonomy was assigned, and abundance was calculated at all possible taxonomic levels. Alpha- and Beta-diversity indices were computed using distinct R packages.
HCC8