Neb next ultra 2 fs dna pcr free library prep kit

Total bacterial DNA was extracted using the TianGen magnetic bead method DNA extraction kit (TianGen, China), following the manufacturer’s instructions. The purity and concentration of the extracted total DNA were assessed using 1% agarose gel electrophoresis. An appropriate volume of sample DNA was placed in a centrifuge tube and diluted to 1 ng/μL with sterile water. The diluted bacterial genomic DNA served as a template for amplifying the V4 hypervariable region of the 16S rRNA gene using primers (515F, 5´-GTGCCAGCMGCCGCGGTAA-3′ and 806R, 5´-GGACTACHVGGGTWTCTAAT-3′). Library construction was performed using the NEB Next^® Ultra™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, China). The library was then quantified by Qubit and Q-PCR. The qualified library was sequenced (PE 250) using the NovaSeq 6,000 platform. Following Reads splicing and filtering, OTUs (Operational Taxonomic Units) clustering, and species annotation, valid data were subjected to species annotation and abundance analysis.

The V3–V4 region of the 16S rRNA were targeted for amplification using specific primers 338F/806R with barcodes (Angebault et al., 2020 (link); Zhang et al., 2021 (link)). PCR amplification was performed with Phanta^® Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China) as described previously (Liu, Zhuang & Wang, 2021 (link)). Sequencing libraries were generated using NEB Next^® Ultra^™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, Ipswich, MA, USA) and quantified by Qubit (Thermo Scientific, Waltham, MA, USA) and Q-PCR. After the library was qualified, paired-end sequencing was performed with a PE250 strategy via the NovaSeq6000 platform (Illumina Inc., San Diego, CA, USA) (Guo et al., 2023 (link)), which was conducted by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).