title: Shotgun metagenomic sequencing of infant gut microbiota
design: Metagenomic DNA was extracted from approximately 100 mg of stool samples using the PowerSoil DNA Isolation Kit (MoBio Laboratories) following the manufacturers protocol with the following modification: samples were lysed by two rounds of two minutes of bead beating at 2.5k oscillations per minute for 2 minutes followed by 1 minute on ice and 2 additional minutes of beadbeating using a Mini-Beadbeater-24 (Biospec Products). DNA was quantified using a Qubit fluorometer dsDNA BR Assay (Invitrogen) and stored at -20?C. Metagenomic DNA was diluted to a concentration of 0.5 ng/L prior to sequencing library preparation. Libraries were prepared using a Nextera DNA Library Prep Kit (Illumina) following the modifications described in Baym et al, 20153. Libraries were purified using the Agencourt AMPure XP system (Beckman Coulter) and quantified using the Quant-iT PicoGreen dsDNA assay (Invitrogen).  For each sequencing lane, 10 nM of approximately 96 samples were pooled three independent times. These pools were quantified using the Qubit dsDNA BR Assay and combined in an equimolar fashion. Samples were submitted for 2150 bp paired-end sequencing on an Illumina NextSeq High-Output platform at the Center for Genome Sciences and Systems Biology at Washington University in St. Louis with a target sequencing depth of 2.5 million reads per sample.
identifiers.namespace: SUB4491864
identifiers.id: N032.E