Zymobiomics microbial community standard

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. .

DNA was isolated from 0.5 mL tomato puree with the FastDNA^® SPIN Kit for Soil (MP Biomedicals, Solon, OH, United States) and following the manufacturers’ protocol using the beat-beating method. To test the efficacy of the protocol in extracting bacterial DNA from tomato puree, selected samples were mixed with 75 μL of thawed mock community suspension (ZymoBIOMICS™ Microbial Community Standard, Zymo Research, United States). In addition, DNA was isolated directly from the pure mock community using 75 μL to provide control DNA samples to test whether there are substances in the DNA extractions from tomato puree, which inhibit PCR. DNA (5 μL) was separated and visually tested for quality by electrophoresis (80 V) on 1% (w/v) agarose gels. In addition, DNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, DE, United States).

Given the low DNA content of bird feces [43 (link)], we were concerned about the influence of environmental microbial contamination in analyzing the sequences [44 (link)]. To understand the sources of contamination, controls were included at the DNA extraction and amplification steps of the sequencing preparation. To evaluate contaminants from the DNA extraction kits, for each kit we included a mock community extraction with 75 μl of bacterial cells from ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA) and a no sample extraction control with only DNA/RNA Shield. To assess contaminants from PCR amplification reagents, for each 96-well plate of PCR amplification, a triplicate mock community amplification with 2 μl of a 1:10 dilution of ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA) and a triplicate no template control reaction. Greater than 99.75% of all reads from ZymoBIOMICS Microbial Community Standards and DNA standards mapped to the expected genera. None of the extraction or no template controls produced quantifiable PCR product and were excluded from further analysis.

Negative and positive DNA extraction controls were extracted and included in the analysis. The negative control was 200 µl RNase free water. Two microbial community standards were used as positive controls; (1) 20 Strain Even Mix Whole Cell Material (ATCC MSA-2002, Virginia, USA) embedded in a stool matrix (#NATROTA-GP, ZeptoMetrix Corporation, New York, USA), and (2) ZymoBIOMICS Microbial Community Standard (#D6300, Zymo Research, California, USA).

DNA was extracted from ~ 0.1 g aliquots of the fecal samples using the NucleoSpin 96 Soil (Macherey–Nagel, Hoerdt, Germany) kit. Bead beating was done horizontally on a Vortex-Genie 2 at 2700 rpm for 2 × 5 min. The samples were almost intact after only 5 min (standard) bead beating; the stool consistency was dry and hard. Double bead beating improved the DNA concentrations of the samples. Two samples did not have sufficient material for re-extraction and were therefore only subjected to 5 min bead beating. A minimum of one negative control was included per batch of samples from the DNA extraction step and throughout the laboratory process (including sequencing). A ZymoBIOMICS Microbial Community Standard (Zymo Research, Freiburg im Breisgau, Germany) was included in the analysis as a positive (mock) control.

The hypervariable region V3-V4 of 16S rRNA gene was sequenced using the Illumina MiSeq platform in the DNA core facility of the University of Helsinki, as described previously^{48 (link)} and in detail in the Supplementary Methods.
To compensate for different quantities of 16S rRNA gene copies in the samples the newborn meconium samples, instrument controls and field controls were preamplified with 21 PCR cycles, mare mouth and vaginal samples with 18 cycles, 24 h foal rectal samples with 16 cycles and 7 d old foal rectal samples and mare fecal samples with 14 cycles. ZymoBIOMICS™ Microbial Community Standard (Zymo Research, USA) was preamplified with 12 cycles.

We extracted DNA from 30 salmon gut mucosa samples. For the 21 parasitized salmon, we also extracted DNA from their associated cestode wash and cestode body samples. In parallel, we extracted four mock community samples as positive controls, using ZymoBIOMICS microbial community standard (Zymo Research) as an input. This mock community comprises eight bacterial strains and two fungal strains that are not expected to be part of the salmon gut microbiome. We also extracted four extraction blanks (sterile water or extraction buffer) as negative controls. The positive and negative controls were all processed in the same manner as the rest of the samples. All extractions were performed using the DNeasy PowerLyzer PowerSoil kit (Qiagen) following the manufacturer’s protocol. Before PCR amplification, we treated each sample with the OneStep PCR inhibitor removal kit (Zymo Research).