Nebnext ultra 2 fs

A total of 20 ng amplified cDNA or circular DNA was used for library preparation using the NEBNext Ultra II FS (New England Biolabs) according to the manufacturer’s protocol. Samples were barcoded using unique dual-index primer pairs (New England Biolabs) and libraries were pooled and sequenced on a HiSeq 4000 instrument (Illumina) or a NovaSeq 6000 instrument with 2× 150-bp paired-end reads for circular DNA libraries and 2× 75-bp paired-end reads for cDNA libraries.

Swabs were transferred into Lysing Matrix E tubes (MP Biomedicals) together with 100 μL of ATL Buffer (Qiagen) containing 1.2% Triton X-100. Next, 10 μl of lysozyme (50 mg/ml, #90,082, Thermo Scientific) were added and incubated at 37 °C for 30 min. Samples were then subjected to bead-beating (FastPrep-24, MP Biomedicals) at 6.0 m/s for 40 s. Samples were centrifuged at 16,000 × g for 5 min. Supernatants were then treated with 10 μl Proteinase K (Qiagen) and incubated at 56 °C for 15 min. DNA was extracted (EZ1 Advanced XL Instrument, Qiagen) using the EZ1 DNA Tissue Kit (Qiagen) and quantified with Qubit dsDNA HS Assay Kit (Life Technologies) and stored at − 20 °C prior to downstream use. The extracted DNA samples were used to construct libraries using NEBNext Ultra II FS (New England Biolabs) and barcoded adaptors according to the manufacturer’s E7805 protocol. Custom indexed primers with 14 PCR cycles were used for enrichment of DNA libraries, which were quantified using Agilent DNA 1000 Kit (Agilent Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). Samples were normalized and pooled for paired-end sequencing (2 × 150 bp) using Illumina HiSeq X platform.