title: Illumina MiSeq sequencing; qiita_ptid_4051:11450.ADEM.145.3651
design: early life wheeze development (asthma vs. controls
library_construction_protocol: DNA isolation of fecal samples was performed according to the benchmarked protocol Q developed by the International Human Microbiome Standards consortium. This method entails a combination of mechanical (repeated bead beating) and chemical lysis followed by column-based purification [PMID: 28967887]. For each DNA isolation batch, additional isolation was performed on PCR-grade water as a negative control. Amplicon libraries and sequencing was performed according by Baseclear [Leiden, the Netherlands] to previously published protocols [REF]. Briefly, the V3-V4 region of the 16S rRNA gene was PCR amplified from each DNA sample in triplicate. Pooled amplicons from the triplicate reactions were purified using AMPure XP purification (Agencourt, Massachusetts, USA) according to the manufacturers instructions and and subsequently quantified by Quant-iT PicoGreen dsDNA reagent kit (Invitrogen, New York, USA). Amplicons were mixed in equimolar concentrations, to ensure equal representation of each sample, and sequenced on an Illumina MiSeq instrument.
attributes.tag: barcode
attributes.value: ATCAGTACTAGG
attributes.tag: center_name
attributes.value: MUMC+
attributes.tag: primer
attributes.value: AATGATACGGCGACCACCGAGATCTACAC TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
attributes.tag: target_gene
attributes.value: 16S rRNA
attributes.tag: target_subfragment
attributes.value: V3-V4