16s metagenomic sequencing library preparation

Libraries for sequencing were prepared according to Illumina’s “16S Metagenomic Sequencing Library Preparation” guide (Part # 15044223 Rev. B), with the exception of using Qiagen HotStar MasterMix for the first PCR (“amplicon PCR”) and halving reagent volumes for the second PCR (“index PCR”). The template specific primers were (without the overhang adapter sequence): 515f (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The first PCR (“amplicon PCR”) was carried out for 25 cycles with annealing temperatures of 55 °C. Diluted pooled samples were loaded on an Illumina MiSeq and sequenced using a 500-cycle MiSeq Reagent Kit v3.

Libraries for sequencing were prepared according to Illumina’s “16 S Metagenomic Sequencing Library Preparation” guide (Part # 15044223 Rev. B), with the exception of using Qiagen HotStar MasterMix for the first PCR (“amplicon PCR”) and halving reagent volumes for the second PCR (“index PCR”). The template specific primers were (without the overhang adapter sequence): 515 f (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′). The first PCR (“amplicon PCR”) was carried out for 25 cycles with annealing temperatures of 55 °C. Diluted pooled samples were loaded on an Illumina MiSeq and sequenced using a 500-cycle MiSeq Reagent Kit v3.