132.PWD.D30.377
DNA was extracted using the QIAamp PowerFecal Pro DNA extraction kit (Qiagen, USA) and assessed via Nanodrop, Qubit, and agarose gel electrophoresis. 16S rRNA amplicon sequencing was performed at the University of Illinois Carver Biotechnology Center (CBC) as follows. 16S amplicon libraries were generated using the Fluidigm Access Array (Fluidigm,USA) amplification according to standard manufacturers protocols. The following primer sequences were used to generate V3-V4 amplicons, following primers: V3_F357_N 5'-CCTACGGGNGGCWGCAG-3 and V4_R805 5-GACTACHVGGGTATCTAATCC-3. The final Fluidigm pools were transferred from the Functional Genomics laboratory to the DNA Services laboratory of the CBC at the University of Illinois at Urbana-Champaign. The final pools were quantitated using Qubit (Life Technologies, Grand Island, NY) and then further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The pool was mixed evenly based on the qPCR values and then denatured and spiked with 20% indexed PhiX control library and loaded onto a MiSeq V2 flowcell at a concentration of 8 pM for cluster formation and sequencing. The libraries were sequenced to a total read length of 250nt. The run generated .bcl files which were converted into demultiplexed compressed fastq files using bcl2fastq v2.20 (Illumina, CA). A secondary pipeline decompressed the fastq files, generated plots with quality scores using FastX Tool Kit, and generated a report with the number of reads per sample/library. The .bcl files were also processed in bcl2fastq v2.20 without demultiplexing, then sorted by initial PCR-specific primer using a custom in-house pipeline.