title: Megatenomic sequencing of human gut microbiome: child fecal sample
design: Fecal samples were homogenized with mortar and pestle under liquid nitrogen. Approximately 50 mg of each homogenized sample was transferred to a 2 mL screw top tube (Axygen, SCT-200-SS-C-S) along with one 3.97 mm steel ball and 250 L of 0.1 mm zirconia/silica beads and (iii) a 500 L mixture of 25:24:1 parts phenol:chloroform:isoamyl alcohol (pH 7.8-8.2), 210 L of 20% SDS, and 500 uL of 2X buffer A (200 mM NaCl, 200 mM Trizma base, 20 mM EDTA) was added to each tube. Samples were then subjected to bead-beating for 4 minutes in a Biospec Minibeadbeater-96, followed by centrifugation at 3220 x g for 4 minutes. A 100 L fraction of the resulting aqueous phase was transferred to a deep 96-well plate together with 70 L isopropanol and 10 L 3M NaOAc, pH 5.5; the solution was subsequently mixed by pipetting 10-times. The crude DNA mixture was chilled at -20 C for approximately 1 hour to precipitate the nucleic acids and then centrifuged at 3,220 x g at 4 C for 15 minutes before removing the supernatant, yielding nucleotide-rich pellets. A Biomek FX robot was used to add 300 L Qiagen Buffer RLT to the pellets and to resuspend the DNA by pipetting up and down 50-times. A 400 L aliqout of the resulting mixture was transferred to an AllPrep 96 DNA plate (Qiagen, Catalog # 80311) which was centrifuged at 3,220 x g for 1 minute at room temperature. DNA was then eluted from the column and retained. DNA extracted from 760 fecal samples was used to prepare shotgun sequencing libraries [reduced-volume protocol based on the Nextera XT (Illumina) strategy. Libraries were quantified, balanced, pooled and sequenced (Illumina NovaSeq 6000, S4 flow cell) to a depth of 2.2 x 107  2.9 x 106 150 nt paired-end reads/sample (mean  SD). DNA reads were demultiplexed (bcl2fastq, Illumina), trimmed to remove low quality bases, and processed to remove read-through adapter sequences Trim Galore v0.6.4. Read pairs where the length of either read was <50 nt after quality and adapter trimming were discarded. The remaining reads were mapped to the human genome (UCSC hg19) using bowtie2 v2.3.4.1 and were filtered to remove H. sapiens sequences.