Smrtbell prep kit 3

High molecular weight (HMW) DNA for single molecule, real‐time (SMRT) sequencing was extracted using the Nanobind CBB kit. High‐fidelity (HiFi) SMRTbell libraries were prepared using the SMRTbell® prep kit 3.0 (PacBio, CA, USA). In summary, 6 μg of HMW DNA from each sample was sheared with hydropores deriving from the Megaruptor 3 DNAFluid+ and the Megaruptor 3 shearing‐kit (Diagenode, MA, USA) to 16–21 kb. Subsequently, DNA damage and fragment ends were repaired, adapters ligated (in some instances employing barcoding) and fragments cleaned. Incomplete SMRTbell templates were removed by a nuclease treatment and purified. All required reagents were included in the SMRTbell® prep kit 3.0 and barcodes were used from the Barcoded overhang adapter kit 8A. Large‐insert SMRTbell libraries for sequencing were achieved by using a size selection with a 10 kb cut‐off using the BluePippin system (SageScience, MA, USA). SMRT sequencing was carried out using the Sequel II/e systems on SMRT Cells 8 M (PacBio, CA, USA). CCS reads were generated and demultiplexing (if required) was performed using standard settings in SMRTLink v11.0 (PacBio, CA, USA), with min passes = 3 and min read quality = 0.99. Generated data for each cell line are summarized in Table S1 and full details on the sequencing runs conducted are given in Table S2.

The purity and quantity of the extracted DNA was assessed using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit 4.0 (Thermo Fisher Scientific, Waltham, MA, USA). For library preparation, the PacBio 16S rRNA degenerate forward and reverse primer set, which is linked with a dual index barcode for demultiplex and sample identification, was used for construction of an indexed library for bacterial 16S rRNA gene (V1 to V9 regions) according to the PacBio standard protocol for the Amplification of bacterial full-length 16S gene with barcoded primers. A total of 14 indexed libraries were pooled together which underwent SMRTbell ligation by utilizing SMRTbell prep kit 3.0 (PacBio, Menlo Park, CA, USA). The SMRTbell ligated library pool was prepared for sequencing by using the Sequel^® II Binding Kit 3.1 (PacBio, Menlo Park, CA, USA) and in 1 SMRT cell for generation of high-fidelity long reads in the Sequel IIe system.

Singular bacteria colonies were picked from CSM agar (pH 6.5) and incubated in 20 mL Tryptic Soy Broth medium in 150 mL baffled shaking flasks at 120 rpm and 28°C overnight. HMW gDNA was extracted according to the instructions of the Quick‐DNA HMW MagBead Kit (Zymo Research).
To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer (Nano Photometer NP80; IMPLEN) and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).
When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 1700g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.

SMRTbell libraries (SMRTbell Prep Kit 3.0.) were prepared from the amplified DNA through blunt ligation following the manufacturer’s instructions (Pacific Biosciences, Menlo Park, CA, USA). Purified SMRTbell libraries from the Zymo (Irvine, CA, USA) and HMP mock communities were sequenced on dedicated PacBio Sequel cells using S/P1-C1.2 sequencing chemistry. Purified SMRTbell libraries from pooled and barcoded samples were sequenced on a single PacBio Sequel cell. Replicate 1 of the samples was sequenced using the S/P2-C2/5.0 sequencing chemistry and replicate 2 of the samples was sequenced using a pre-release version of S/P3-C3/5.0 sequencing chemistry. Amplicon sequencing was performed by Shanghai Biozeron Biotechnology Co., Ltd. (Shanghai, China).