cystic fibrosis gut microbiome 16s amplicon sequencing
Construction protocol: The v4-v5 hypervariable regions of the bacterial SSU rRNA gene were amplified using degenerate primers. The primers were 518F (CCAGCAGCYGCGGTAAN) and 926R (CCGTCAATTCNTTTRAGT CCGTCAATTTCTTTGAGT CCGTCTATTCCTTTGANT).  Amplification was done with fusion primers containing the 16S-only sequences fused to Illumina adapters. The forward primers included 4 random bases followed by a 5 nt multiplexing barcode 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.
SUB5304786
110_L_STOOL_Bv4v5