Hm 782d

To determine the effects of different primer sets, and different DNA extraction procedures on sequencing results, genomic DNA from Microbial Mock Community B (Even, Low Concentration), v5.1L, for 16S rRNA Gene Sequencing (HM-782D), and cells from Microbial Mock Community C in phosphate buffered saline (PBS) (HM-280) and in PBS and 40 % Glycerol (HM-281) were obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project (Manassas, VA). Mock community DNA was used as template DNA for sequencing using 3 primer sets, per platform, as outlined below.

All ileal digesta and fecal samples obtained at postmortem were subjected to 16S rRNA gene metabarcoding targeting the V3 hypervariable region, as described previously (30 (link)). DNA concentrations of the purified libraries were then measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, United Kingdom) using a Qubit double-stranded DNA high-sensitivity assay kit (Thermo Fisher Scientific). Using the readings obtained by the Qubit instrument, four library pools were constructed using equimolar concentrations of DNA from each sample. A reagent-only control and mock bacterial community (HM-782D; BEI Resources, ATCC, Manassas, VA) were included as part of each sequencing run to assess background contamination, sequencing error rate, and inter-run variability. On submission to the sequencing center (Edinburgh Genomics, United Kingdom), library pools were quantified using a Quant-iT PicoGreen double-stranded DNA assay kit (Thermo Fisher Scientific) to ensure a sufficient yield for sequencing. Sequencing was carried out using an Illumina MiSeq platform (Illumina, USA), using V2 chemistry and producing 250-bp paired-end reads. The generated sequences (with primers removed) are available publicly through the European Nucleotide Archive (ENA) under accession number PRJEB33396.

Stool specimens were thawed at 4 °C and were diluted 1:3 with milliQ water to create a stool slurry. DNA was isolated using the ZR-96 Fecal DNA Kit (Zymo Research, Irvine, CA) following a validated protocol [20 ]. The Illumina MiSeq sequencing library preparation protocol for 16S rRNA gene amplicons was followed with modifications [20 ]. Briefly, the 16S rRNA V4 region was amplified using primers 515fXT (GTGBCAGCMGCCGCGGTAA) and 806rXT (GGACTACHVGGGTWTCTAAT). Quality control, quantification, normalization, pooling, and sequencing of the library was performed as previously described [20 ]. Approximately 11 pM of the pooled, multiplexed samples were mixed with 37.5% PhiX spike-in control DNA and sequenced on an Illumina MiSeq instrument to generate 2 × 300 bp reads. Four MiSeq runs were performed with 56 multiplexed samples per run. In total, 224 samples were sequenced including technical replicates, mock communities of known composition (HM-782D; BEI Resources, Manassas, VA) and no-template controls.

We amplified the V4V5 region of bacterial 16S rRNA genes in wastewater samples using primers 518F and 926R [28 (link)]. The following setup was used: 12.5-l 2 KAPA HiFi HotStart ReadyMix PCR (Roche), 1.5-l of each 5-M forward and reverse primer working solutions, 7.5-l sterile water, and 2-l 100-diluted DNA template. PCR was run on a vapo-protect Mastercycler pro S (Eppendorf) under the following conditions: 95 C for 5 min; 22 cycles of 98 C for 20 s, 55 C for 15 s, 72 C for 1 min; 72 C for 1 min; 4 C hold. We included one negative control and one mock community (#HM-782D, BEI Resources). Triplicate PCRs were pooled and cleaned with Agencourt AMPure XP beads (Beckman Coulter), following the manufacturers protocol. Sample libraries were prepared according to the Illumina MiSeq protocol in the Nextera XT Index kit (Illumina). Indexed PCR amplicons were cleaned with AMPure beads and normalized with the SequalPrep Kit (ThermoFisher Scientific). Sequencing was carried out on an Illumina MiSeq with 2 250 chemistry at the Great Lakes Genomics Center (greatlakesgenomics.uwm.edu).

To assess the quality of the extraction, PCR and sequencing procedures, one pigeon sample (#13) was duplicated by tagging each of two amplicons obtained from this sample with a unique pair of barcodes. The β diversity between the sequences obtained from these samples, measured using the weighted UniFrac metric [28 (link)], was 0.054. Further, 5 amplicons were generated in a separate PCR from DNA extracted from a synthetic bacterial population (BEI Resources, Manassas, VA, cat no. HM-782D). The ten weighted UniFrac distances between these 5 samples were 0.033, 0.036, 0.018, 0.036, 0.027, 0.035, 0.031, 0.028, 0.030 and 0.025. The mean of these distance values equals 0.03 and represents 4.9% of the average. The UniFrac distance was 0.61 between the 46 bird samples analyzed here (1035 pairwise distance values). The mean phylum-level proportion for these five control samples was 0.041 (SD = 0.001) for Deinococcus-Thermus, 0.0433246 (SD = 0.002) for Actinobacteria, 0.37 (SD = 0.006) for Proteobacteria, 0.488 (SD = 0.008) for Firmicutes, 0.048 (SD = 0.003) for Bacteroidetes and 0.005 (SD = 0.002) for unclassified bacteria. The expected proportions for the same 6 phyla were 0.05, 0.10, 0.30, 0.50, 0.05 and 0, respectively.