Zymobiomics dna kit

Bacterial genomic DNA samples were extracted using ZymoBIOMICS DNA Kits (Zymo Research, CA, USA), following the manufacturer’s instruction. The PCR reaction mixture (25 µL) contained a 1× JumpStart REDTaq ReadyMix (Sigma) and each primer (Table 2). The list of primers used in the mPCR are also presented in Table 2 [34 (link),35 (link),36 (link),37 (link)]. The PCR reaction was performed as follows: initial activation of DNA polymerase at 95 °C for 3 min, plus 35 cycles of denaturation at 95 °C for 30 sec, primer annealing and extension at 62.5 °C for 1.30 min, and a final extension at 72 °C for 5 min. A negative control was included in each run, consisting of the same reaction mixture but with water instead of template DNA.
The PCR products (5 µL) were analyzed using gel electrophoresis on 1.5% (w/v) agarose gel in 0.5 × TBE buffer at a constant voltage of 100 V for 30 min (Mupid exU system; Takara; Tokyo, Japan). The gels were stained with ethidium bromide and visualized under ultraviolet light (GeneGenius Bioimaging System; SynGene; Cambridge, UK). The sizes of the PCR products were determined by comparison with molecular-sized standards (GeneRuler™ 100 bp Plus DNA ladder; Thermo Fisher Scientific; Vilnius, Lithuania).

Oocysts of C. cayetanensis (preserved in 2.5% potassium dichromate solution) obtained from a laboratory strain collection and environmental (fruit and soil) samples were subjected to DNA extraction using the ZymoBIOMICS DNA Kit (Zymo Research, Irvine, CA, USA) following manufacturer’s instructions. Purified DNA was diluted to reach a concentration of 1 ng/µL and stored at −20 °C.

DNA from microbial colonies for Sanger sequencing or cells grown in the enrichment medium for high-throughput sequencing was isolated using ZymoBIOMICS DNA Kit (Zymo Research, Irvine, CA, USA).

Total genomic DNA from the collected samples was extracted using a ZymoBIOMICS DNA kit (Zymo Research, Irvine, CA, USA). To prepare SSU rRNA gene amplicon libraries we used the Quick-16S NGS Library Prep Kit (Zymo Research), according to the manufacturer’s protocol for amplification and barcoding. Both sets of included primers (V1–V2 and V3–V4) were used to generate amplicon libraries. Negative controls (no DNA template and blank DNA extractions) were included. Bidirectional sequencing of all samples together was performed on a MiSeq instrument (Illumina, San Diego, CA, USA) using a v3 600 cycles kit, according to the manufacturer’s instructions.

DNA was recovered from filters using the ZymoBiomics DNA Kit (Zymo Research, Costa Mesa, CA, United States), according to manufacturer’s guidelines. Extracted DNA was eluted into 100 μL of buffer and aliquots were stored at −20°C until analyzed with droplet digital PCR (ddPCR) on the Bio-Rad QX200 platform (Bio-Rad, Hercules, CA, United States). DNA from a halophilic, alkaliphilic archaeon (Natronomonas pharaonis) was added to the lysis buffer prior to extraction as an external extraction and inhibition control following USEPA method 1611 guidelines (USEPA, 2012 (link)). Negative extraction controls (NEC) containing only lysis buffer and halophile DNA were processed alongside each set of extracted samples.
Human-associated genetic markers and Enterococcus were quantified using digital PCR. Primer and probe sequences were those from published methods for detection of the HF183 (Cao et al., 2015 (link)) and Lachno3 (Feng et al., 2018 (link)) human-associated markers and for detection of Enterococcus (Cao et al., 2015 (link)). Primer and probe sequences are included in the Supplementary Material (Supplementary Table 2) and detailed methods employed have been described previously (Steele et al., 2018 (link)).