Metagenomic sequencing of human stool
DNA (10ng to 500 ng) was sheared into fragments of approximately 300-400bp in a Covaris E210 system (96 well format, Covaris, Inc., Woburn, MA) followed by purification of the fragmented DNA using AMPure XP beads. DNA end repair, 3-adenylation, ligation to Illumina multiplexing PE adaptors, and ligation-mediated PCR (LM-PCR) were all completed using automated processes.KAPA HiFi polymerase (KAPA Biosystems Inc.) was utilized for PCR amplification (6-10 cycles), which is known to amplify high GC and low AT rich regions at greater efficiency. A Fragment Analyzer (Advanced Analytical Technologies, Inc.) electrophoresis system was used for library quantification and size estimation. Prepared libraries were then pooled and sequenced on the Illumina HiSeq2000 platform.