Sequelprep normalization plate kit

Library prepared as previously explained in Inoue et al., 2016 [64 (link)], and deep sequencing were performed using a MiSeq apparatus (Illumina K.K., Tokyo, Japan). Specifically, 341F and 805R primers with 5′ overhang adapter sequences for the second PCR (Polymerase Chain Reaction) were used to amplify the V3–4 region of 16S rRNA genes in each sample. NucleoFast 96 PCR plates (TaKaRa bio, Shiga, Japan) were used to purify the amplicons, and a unique combination of dual indices (I5 and I7 indices) was attached in the second round of PCR. After purification using a SequelPrep Normalization Plate Kit (Thermo Fisher, Tokyo, Japan), the concentration of each sample was normalized. Next, the samples were pooled and concentrated using AMPure XP beads (Beckman Coulter, Tokyo, Japan). Through the SequelPrep Normalization Plate Kit (Thermo Fisher, Tokyo, Japan), ten pM of the library combined with 20% phiX Control (Illumina) was sequenced with 285 bp paired-end bases on MiSeq.

DNA was extracted using the MoBio PowerSoil^® DNA Isolation kit (Qiagen, Germantown, MD, United States) and quantified using the PicoGreen fluorometric assay (Thermo Fisher Scientific, Waltham, MA, United States). A 406 bp region of the RNA polymerase gene (rpoB) was amplified by PCR (∼25 ng DNA in a 25 μl reaction) using Streptomyces-specific primers Smyces_rpoB1563F and Smyces_rpoB1968R as described elsewhere (Higgins et al., 2021 (link)). The PCR reactions consisted of 25 ng DNA, 12.5 μl of Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, United States), 0.625 μl of 4X Quant-iT PicoGreen dsDNA assay reagent (Thermo Fisher Scientific, Waltham, MA, United States), and 1.25 μl each of 10 μM dual-barcoded forward and reverse primers modified for Illumina sequencing as described in Kozich et al. (2013) (link). PCR products from triplicate reactions were pooled and normalized using the SequelPrep Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA, United States). Fragments of 450 bp in length were size selected with a 1% agarose gel and subsequently extracted and purified from the gel band. Pooled samples were concentrated to 2 ng/μl using a vacuum concentrator and sequenced on an Illumina MiSeq instrument (2 × 300 bp) at the Biotechnology Resource Center, Cornell University.