Kapa hifi hotstart ready mix

For 16S rRNA amplification, 12.5 ng of DNA was amplified using 0.2 μM of both forward primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, Metabion) and reverse primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, Metabion).
KAPA HiFi HotStart Ready Mix (KK2601; KAPABiosystems) was used for the PCR amplification. PCR was performed using a first denaturation of 95 °C for 3 min (min), followed by 25 cycles of amplification at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, final elongation at 72 °C for 5 min and the amplified DNA was stored at 4 °C. DNA gel electrophoresis of all the samples was performed to verify the amplicon specificity.
Further, samples were then purified (Agencourt AMPure XP, Beckman Coulter) and PCR amplicons were indexed using Nextera XT index and KAPA HiFi HotStart ReadyMix. PCR was performed using a first denaturation of 95 °C for 3 min, followed by 8 cycles of amplification at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30, final elongation at 72 °C for 5 min. Samples purified were then validated using BioAnalyzer (Bioanalyzer DNA 1000, Agilent) and 4 nM of each library pooled using unique indices before sequencing on a MiSeq (Illumina) and paired 300-bp reads.

The DNA libraries are prepared using the Thru PLEX Tag-Seq HV kit (Takara Bio Inc. Japan) as per the manufacturer’s instructions. The hybrid capture is performed using IDT xGen Hybridization and Wash Kit (IDT Inc. USA) protocol. Streptavidin-coated magnetic beads are used to bind and extract biotinylated probe-hybridized target DNA. This protocol includes target enrichment by PCR prior to sequencing using KAPA HiFi HotStart Ready Mix and DNA clean up using Agencourt AMPure XP PCR Purification system (Beckman Coulter, USA). The capture library products are assessed using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer High Sensitivity DNA assay (Agilent Technologies Inc., USA).