Zymobiomics dna microprep kit

Bacterial DNA extraction from seminal samples and mock community was performed with ZymoBIOMICS DNA Microprep kit (Zymo Research, Irvine, CA, USA). To allow the release of DNA, a bead beating in lysis solution was performed to lyse cell walls and membranes. Then, slight modifications were included into the manufacturer’s protocol, to filter samples. All supernatant was filtered in Zymo-Spin III-F by centrifugation at 8000× g for 1 min and washing repeatedly in a Zymo-Spin IC-Z column to purify DNA before elution. Sterile gloves and a horizontal laminar flow cabinet, previously sterilized with DNA-degrading products, and UV irradiation was used during extraction step. A sterile swab was placed inside the cabinet as a negative environmental control and a blank control was included to observe possible kitoma contamination.

ZymoBIOMICS DNA Microprep Kit (Zymo Research, Irvine, CA, USA) was used for DNA extraction from the biopsy specimens, per the manufacturer’s protocol. PCR amplification of the V4 region of the 16S ribosomal RNA gene was followed by 250 × 2 paired-end sequencing on an Illumina HiSeq (Illumina, San Diego, CA, USA), as previously described [32 (link),34 (link)]. The sequences were processed using the DADA2 pipeline in R, and SILVA 132 database was used for taxonomy assignment [35 (link)]. Next, data were incorporated into QIIME 2 version 2019.10 [36 (link)]. Amplicon sequence variants were filtered out if they were not present in at least 15% of all samples (3,449,114/4,777,389 sequences were kept after this filtering step). Sequence depths ranged between 9,030 and 246,235 per sample with a median value of 71,618.

DNA was extracted using a ZymoBiomics DNA Micro Prep kit, and RNA was extracted using a ZymoBiomics RNA Mini Prep kit according to the manufacturer’s specifications (Zymo Research, Irvine, CA, USA). At each site, triplicate sediment samples were collected for a total of 63 samples. All three samples from each site were used for 16S analysis, while only two were used for metatranscriptomics. For sediment samples, 0.25 g of sediment was used as input. A single water sample was collected from each site by pushing 200 to 600 mL of water through a 0.2-μm membrane filter (Millipore Sigma, Burlington, MA, USA). The entire membrane was then used as input for extraction. The V4 region of the 16S rRNA gene was amplified using primers and conditions previously described (60 (link)), and the libraries were prepared as previously described (23 (link)). See Text S1 for primers, thermocycler conditions, library preparation, and sequencing conditions. Data generated from sequencing were downloaded and imported into QIIME2-2019.7 (61 (link)) for preprocessing, diversity, and biomarker analysis (see Text S1).

DNA was extracted using a ZymoBiomics DNA Micro Prep kit, and RNA was extracted using a ZymoBiomics RNA Mini Prep kit according to the manufacturer’s specifications (Zymo Research, Irvine, CA, USA). At each site, triplicate sediment samples were collected for a total of 63 samples. All three samples from each site were used for 16S analysis, while only two were used for metatranscriptomics. For sediment samples, 0.25 g of sediment was used as input. A single water sample was collected from each site by pushing 200 to 600 mL of water through a 0.2-μm membrane filter (Millipore Sigma, Burlington, MA, USA). The entire membrane was then used as input for extraction. The V4 region of the 16S rRNA gene was amplified using primers and conditions previously described (60 (link)), and the libraries were prepared as previously described (23 (link)). See Text S1 for primers, thermocycler conditions, library preparation, and sequencing conditions. Data generated from sequencing were downloaded and imported into QIIME2-2019.7 (61 (link)) for preprocessing, diversity, and biomarker analysis (see Text S1).