2.2. Fecal Sample Collection, DNA Extraction, Amplicon Generation, and 16S rRNA Sequencing
diarrhea
COLD, INFECTIOUS DISEASE
Fecal samples were routinely collected at baseline (3 months) as well as at 6, 9, 12, 18, and 24 months for enteric pathogens, ensuring the absence of diarrhea in the preceding 72 h. Once the subject was identified by the field teams in their weekly visits, a stool kit (comprising of diaper, spoon, container, and gloves) was provided to the family of the subject to collect a fresh fecal sample. A significant portion of the fecal sample was placed in the plastic screw-capped container using the disposable plastic spoon immediately after collection, ensuring the collection only of specimens not in contact with the diaper lining.The collected fecal samples were transported in a special cold storage box with cold chain maintenance at a temperature of 2–8 °C within 6 h of collection from the field site to the Infectious Disease Research Laboratory (IDRL) at the Department of Pediatrics, Aga Khan University. In the research lab, these stool samples were stored at −80 °C. Ten children representing each of the three groups, from both urban and rural sites, and with fecal samples available at all of the time points (3, 6, 9, 12, 18, and 24 months), were randomly chosen for this sub-study. This provided a total of 360 fecal samples for the assessment of intestinal microbiota.DNA was extracted from stool samples in the IDRL using a QIAamp Stool Mini kit (Qiagen, Inc., Valencia, CA, USA) and shipped to Tufts Medical Center in Boston. Upon receipt at Tufts Medical Center, DNA concentration was measured using a Nanodrop ND-1000 (Thermo Scientific, Wilmington, DE, USA) and normalized to 50 µg/mL in all samples by the addition of UltraClean PCR Water (MoBio, Thermo Fisher Scientific, Waltham, MA, USA). For sequencing runs performed on LR70 plates, (Roche, Indianapolis, IN, USA). PCR primers were designed to amplify the V4 hypervariable region of the genes for the 16S rRNA gene of both Bacteria and Archaea. The forward primer contained the V4 specific sequence AYTGGGYDTAAAGNG, an octamer barcode [PCR reactions were carried out in triplicate in parallel with a barcode-specific negative control; reactions yielding no amplicons or those in which the negative controls amplified were repeated. DNA concentration of amplicons was determined using a Quant-iT assay (Invitrogen, Carlsbad, CA, USA) and they were then pooled in equimolar concentrations. The amplicon pool was purified twice using an AMPure XP kit (Agencourt, Indianapolis, IN, USA) and sequenced on a Roche 454 Genome Sequencer GS FLX using the LR70 platform according to Roche protocols at the Virginia Bioinformatics Institute, and subsequently at the Tufts University Core Facility Genomics Core using a Roche 454 Genome Sequencer GS FLX+ and the Lib-L Sequencing kit according to Roche protocols. Replicates of the same sample were sequenced on both systems and the results showed no significant differences.