title: LMIC human infant stool sample 2854, whole-genome shotgun metagenomic sequencing to validate qPCR Metagenome
design: Depending on the sample, 20 to 1,500 ng of DNA extracted from fecal samples was sheared in a 50 L screwcap microtube using a Covaris M220 instrument (Covaris, Woburn, MA) at 50 peak power, 20 duty factor, 20C, 200 cycles per burst and 25-31 seconds duration. Illumina sequencing libraries were prepared using the KAPA High-Throughput Library Preparation Kit with Standard PCR Amplification Module (Kapa Biosystems, Wilmington, MA), following the manufacturers instructions except for the following changes: libraries were prepared using 1/4th volumes for all steps except size selected to ~5001500 bp following double-sided size selection protocols with modified volumes of 41 l PEG/NaCL solution and 5ul AMPure XP beads. Indexing of each individual sample was done using standard desalted TruSeq HT dual index adapters from Integrated DNA Technologies (Coralville, IA) at 0.75 M final concentration, and a total of 3-18 PCR cycles were used to minimize bias. Libraries were quantified using the KAPA Library Quantification Kit, except with 10 l volume and 90 s annealing/extension PCR, and then pooled and normalized to 4 nM. Pooled libraries were re-quantified by ddPCR on a QX200 system (Bio-Rad, Hercules, CA), using the Illumina TruSeq ddPCR Library Quantification Kit and following the manufacturers protocols, except with an extended 2-min annealing/extension time. Libraries were sequenced on an Illumina MiSeq instrument (v2 500-cycle kit; Illumina, San Diego, CA) per the manufacturers protocols.