Microbial Community Profiling via Illumina Sequencing

Library Preparation and Sequencing: The bacterial community composition was determined by Illumina NextSeq-based high-throughput sequencing (HTS) of the 16S rRNA gene V3-region, according to Krych and colleagues. (2018) [46 (link)] and is described in detail in the supplementary materials and methods. Briefly, the amplified fragments with adapters and tags were purified and normalized using custom made beads, pooled, and subjected to 150 bp pair-ended Illumina NextSeq (V3 region 16S rRNA) sequencing. The raw dataset containing pair-end reads with corresponding quality scores were merged and trimmed, followed by de-replication, purging of chimeric reads, and constructing high quality (97% similarity level) Operational Toxonomic Unit (OTU) that and taxonomically assigned using sintax coupled to the EZtaxon 16S rRNA gene reference database. The sequencing dept. was on average 68,194 read per sample before filtering going down to 62,426 after filtering.
Sequencing data pre-processing: The dataset was purged for OTU’s which were detected below 0.005% across all samples, and normalization was accomplished using MetagenomeSeq v 1.32.0 based on Cumulative Sum Scaling algorithm [47 (link)].