CHILD Cohort Study ITS2

CHILD Study participantsThis project significantly expands the understanding of the early life gut mycobiome within participants in the CHILD Study by providing a novel and independent measure of microbiota changes by analyzing the mycobial component. This multi-center longitudinal, prospective, general population birth cohort study follows infants from pregnancy to age 5 years and beyond. Briefly, enrollment for CHILD began in 2008 and closed in 2012, including a total of 3,621 pregnant people from four cities (Vancouver, Edmonton, Winnipeg, Toronto) across Canada enrolled along with eligible infants (n=3,455) who had no congenital abnormalities and were born at a minimum of 34 weeks of gestation. Informed consent was obtained from parents during this study, and detailed information was collected using a combination of questionnaires and in-person clinical assessments.Sample collection and storageStool samples were collected from diapers at a home visit at a mean (SD) age of 3.8 (1.1) months and at a clinic visit at 12.5 (1.6) months. Samples were briefly refrigerated (4 degrees C) and then aliquoted into four 2 mL cryovials using a stainless-steel depyrogenated spatula. They were subsequently frozen at -80 degrees C, and the time from collection to long-term storage was recorded for statistical adjustment.DNA extraction, library preparation, and ITS2 amplicon sequencingDNA extraction, library preparation, and ITS2 amplicon sequencing of fungal rDNA from stool were performed by Diversigen (Minneapolis, MN, USA). DNA was extracted using the MO Bio PowerFecal (Qiagen) kit automated on a QIAcube (Qiagen) with bead beating in 0.1 mm glass bead plates. Fungal DNA was quantified by qPCR targeting the ITS2 region (5.8SR/ITS4 primers) between the 5.8S and 26S rRNA subunits. Library preparation followed a protocol derived from Gohl et al., 2016, using KAPA HiFi Polymerase for amplification of the ITS2 region. Libraries were sequenced on an Illumina MiSeq using paired-end 2 x 250 reads with the MiSeq Reagent Kit v3.