16S Metagenomics of Bombyx mori: D119 3-1
Libraries were prepared based on the 16S Metagenomic Sequencing Library Preparation guide by Illumina. The amplicon PCR mix was prepared using 16S V3-V4 PCR forward and reverse primers containing 5 overhang sequences. Pairs of different index 1 and index 2 from the Nextera XT Index kit were selected such that each sample would have a unique pair of indices. A round of clean-up was performed using AMPure XP beads after 16S PCR and Index PCR. Calculated library concentrations were then normalized to the lowest concentration (nM) among all libraries using nuclease-free water. Each diluted library was then pooled. An appropriate volume of 0.2 N NaOH was used to denature the pooled library. An appropriate volume of pre-chilled HT1 was then added to the denatured library to produce a 20 pM denatured library. This was then diluted to an 8pM denatured library and combined with a similarly prepared PhiX library such that at least 15% PhiX was spiked in.