alias: UC20021408Mv6_ill_merged
title: Illumina v6 sequencing ulcerative colitis microbiome study: Sample UC20021408M
design: "Bacterial v6 Illumina amplicons: The v6 hypervariable region of the SSU rRNA gene was amplified using degenerate primer sets. The primers were 967F (CNACGCGAAGAACCTTANC; CTAACCGANGAACCTYACC; CAACGCGMARAACCTTACC; ATACGCGARGAACCTTACC) and 1064R (CGACRRCCATGCANCACCT).  Amplification was done with fusion primers containing the 16S-only sequences fused to Illumina adapters. The forward primers included 5 nt multiplexing barcodes and the reverse a 6 nt index. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 10 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix (ThermoFisher), 3 mM MgSO4 and 0.2 uM of each primer. We added approximately 10-25 ng template DNA to each PCR and ran a no-template control for each primer pair. Amplification conditions were: initial 94C, 3 minute denaturation step; 30 cycles of 94C for 30s, 57C for 45s, and 72C for 60s; final 2 minute extension at 72C. The triplicate PCR reactions were pooled after amplification and purified using Ampure followed by PicoGreen quantitation and Ampure size selection.  Raw fastq have not been trimmed of inline barcodes or 16s primers. Merged_unique.fastq.gz files contain the trimmed, merged, unique sequence only, where no mismatches were allowed. The definition line indicates the frequency of each unique sequence. "
identifiers.namespace: MBL-phs000262
identifiers.id: UC20021408Mv6_ill_merged