16S Amplicon Sequencing of Gut Microbiome

Fecal samples were aliquoted and immediately stored at −80 C. DNA was extracted from 0.1 gram feces using the Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (ZymoResearch, CA, USA) according to manufacturer instructions with minor adaptations, as described previously.^{41 (link)} Quality control, library preparation and sequencing were performed by GenomeScan B.V. (Leiden, The Netherlands) using the NEXTflex™ 16S V4 Amplicon-Seq Kit (BiooScientific, TX, USA) and the Illumina NovaSeq6000 platform (paired-end, 150bp). Raw read processing was performed using the NG-Tax 0.4 pipeline with following settings: forward read length of 120, reverse read length of 120, ratio OTU abundance of 2.0, classify ratio of 0.9, minimum threshold of 1*10⁻⁷, identity level of 100% and error correction of 98.5, using the Silva_132_SSU Ref database.^{41–43 (link)} The obtained OTU table was filtered for OTUs with less than 0.005% relative abundance.^{44 (link)} As quality controls for both DNA extraction and sequencing, we included ZymoBiomics Microbial Community Standard (Zymo Research, Irvine, California, USA) ZymoBiomics Microbial Community DNA Standard (Zymo Research) and three negative DNA extraction controls. Raw sequencing data are available at ENA (https://www.ebi.ac.uk/ena) under accession number PRJEB36316. All analytical R code can be found at https://github.com/qducarmon/hookworm_microbiota_16S. .