Kapa hifi hotstart pcr kit

Using the MiSeq system (Illumina, San Diego, CA, USA), 16S rRNA gene sequencing was performed as described previously^{32 (link)}. The V3–V4 region of the bacterial 16S ribosomal RNA gene was amplified using the KAPA HiFi HotStart PCR kit (Kapa Biosystems, Woburn, MA, USA) and barcode-indexed primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′). The amplicons were purified using AMpureXP (Beckman Coulter, Tokyo, Japan) and quantified using Qubit (Thermo Fisher Scientific, Waltham, MA, USA). The purified amplicons were pooled in equimolar concentrations (1.20 ng/μL) and sequenced.

On-target and potential off-target mutations were amplified using a KAPA HiFi HotStart PCR Kit (#KK2501; KAPA Biosystems, Wilmington, MA, United States) for deep sequencing library generation. Pooled PCR amplicons were sequenced using the MiSeq with TruSeq HT Dual Index system (Illumina, San Diego, CA, United States).

The resulting PCR amplicons were diluted up to 10 ng/μL and then used as templates within the second-step PCR for further amplification, and to include the indexes (barcodes) as well as the Illumina adaptors. The combinatorial use of index primers resulted in unique samples that were pooled and sequenced on one Illumina MiSeq run. In more detail, amplification reaction was performed using the KAPA HiFi HotStart PCR Kit in a final volume of 50 μL. Each reaction contained 10 μL of KAPA HiFi Fidelity Buffer (5×), 1.5 μL of dNTPs solution (10 mM each), 5 μL of the forward indexing primer (10 μM), 5 μL of the reverse indexing primer (10 μΜ), 1 μL of KAPA HiFi HotStart DNA Polymerase (1 U/μL), 2 μL from the diluted PCR product (10 ng/μL) and 25.5 μL of sterile deionized water. The PCR amplifications were performed with a 3-min incubation at 95 °C followed by 8 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and a final 5-min terminator reaction at 72 °C. The resulting amplicons from indexing PCR were purified using Macherey-Nagel’s NucleoMag^® NGS Clean-up and Size Selection kit according to the manufacturer’s recommendations. Amplicons from different samples were quantified with a Quawell Q5000 micro-volume UV−Vis spectrophotometer and merged in equimolar ratios (8 nM).