Metagenomic analysis of viruses in the fecal micorobiota of monozygotic twins and their mothers
Amplification of bacterial 16S rRNA genes consists of amplicons ofapproximately 330bp, spanning variable region 2 (V2) of bacterial 16 rRNA genes that were generated by using PCR and which contained a modified primer 8F which contains 454 primer B and bacterial universal primer 8F and a modified primer 338R that contains 454 primer A, a sample specific barcode, and the bacterial primer 338R. Four replicate polymerase chain reactions were performed for each pooled fecal DNA sample. Each 20 microliter reaction contained 100ng of gel purified DNA (Qiaquick, Qiagen). Replicate PCRs were subsequently pooled and purified using Ampure magnetic purification beads (Agencourt). DNA was quantified using Picogreen (Invitrogen) and an equimolar amount of each sample was used for multiplex 454 FLX amplicon pyrosequencing.