Hotstartaq plus master mix kit

The DNA of sediment samples was extracted using E.Z.N.A Soil DNA kit (OMEGA, United States) according to the manufacturer protocol and stored at −20°C until further analysis. DNA extracts were checked for quantity and purity with NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific) and subsequently submitted to the sequencing platform Mr. DNA (Molecular Research LP, Shallowater, TX). The 16S ribosomal RNA (rRNA) gene V4 variable region PCR primers 515/806 for Eubacteria and Archaea with barcode on the forward primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, United States) under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. PCR products were used to prepare a DNA library following Illumina TruSeq DNA library preparation protocol. Sequencing was performed on Illumina MiSeq Next Generation Sequencer in accordance with the manufacturer guidelines.

The V6–V8 region of the 16S rRNA gene was amplified using universal (degenerate) PCR primers 799F (acCMGGATTAGATACCCKG) and bac1193R (CRTCCMCACCTTCCTC). Amplification was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD, USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, with a final elongation step at 72 °C for 5 min. PCR products were confirmed using a 2% agarose gel. PCR products were then used to prepare DNA libraries following Illumina MiSeq DNA library preparation protocol. Sequencing was performed at the University of Arizona Genetics Core (UAGC) on a MiSeq following the manufacturer’s guidelines.

Library preparations and sequencing were performed by a commercial service (Molecular Research Laboratory, Shallowater, TX, United States). The 16S rRNA gene variable region V4 (Gray et al., 1984 (link)) was amplified using Illumina (San Diego, CA, United States) barcoded oligonucleotides that contain the priming sequences 515F-GTGYCAGCMGCCGCGGTAA (Parada et al., 2016 (link)) and 806R-GGACTACNVGGGTWTCTAAT (Apprill et al., 2015 (link)). Polymerase chain reaction (PCR) was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany). The thermocycling protocol was as follows: polymerase activation by heating at 94°C for 3 min; 28 cycles of melting at 94°C for 30 s; annealing at 53°C for 40 s; and primer extension at 72°C for 1 min. An additional elongation step of 72°C for 5 min was added to the last cycle. After 2% agarose gel electrophoresis, successfully produced amplicons were pooled in equal molar amounts and purified using Ampure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN, United States). The library was sequenced with an Illumina MiSeq using the manufacturer’s protocol. After sequencing, barcodes were removed. Sequences shorter than 150 pb were purged, and chimeras were removed. Ten of ten samples were successfully sequenced from the winter samples while nine of ten samples were successfully sequenced from the summer samples.

All 120 raw milk samples collected during summer and winter were thawed on ice, 10 mL milk was centrifuged at 5500g for 20 min at 4 °C and the pellet were used for gDNA extraction using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. Genomic DNA was sent for Next-generation sequencing (NGS) at Molecular Research DNA (MRDNA, Shallowater, TX, USA). The hypervariable region V4 of the the16S rRNA gene was chosen to study the microbial profile. The universal primers 515/806 were used to amplify the V4 region in a 30-cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) using the following conditions: 95 °C for 5 min, followed by 30–35 cycles of 95 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 10 min was performed. Later, 2% agarose gel was used to assess the amplification success and intensity of the bands. Multiple samples were pooled together in equal proportions based on their DNA concentrations and molecular weight, purified using calibrated Ampure XP beads, and the Illumina DNA library was prepared. Sequencing was performed on a MiSeq (MR DNA) following the manufacturer's guidelines, generating 250 bp paired-end (PE) reads.

Sequence libraries were constructed using the HotStarTaq Plus Master Mix Kit (Qiagen Ltd., UK) and a one-step PCR with 30 cycles. Bacterial tag encoded amplicon pyrosequencing was carried at the Research and Testing Laboratory (Lubbock, TX) using a Roche 454 FLX instrument with titanium reagents.