16S rRNA Gene Sequencing of Biopsy Specimens

ZymoBIOMICS DNA Microprep Kit (Zymo Research, Irvine, CA, USA) was used for DNA extraction from the biopsy specimens, per the manufacturer’s protocol. PCR amplification of the V4 region of the 16S ribosomal RNA gene was followed by 250 × 2 paired-end sequencing on an Illumina HiSeq (Illumina, San Diego, CA, USA), as previously described [32 (link),34 (link)]. The sequences were processed using the DADA2 pipeline in R, and SILVA 132 database was used for taxonomy assignment [35 (link)]. Next, data were incorporated into QIIME 2 version 2019.10 [36 (link)]. Amplicon sequence variants were filtered out if they were not present in at least 15% of all samples (3,449,114/4,777,389 sequences were kept after this filtering step). Sequence depths ranged between 9,030 and 246,235 per sample with a median value of 71,618.