Smrtbell template prep kit 1.0 spv3

The microbial DNA was retrieved using a PowerSoil DNA Isolation Kit (QIAGEN) according to the supplier’s protocol with slight modifications as described below. The filters were removed from the container, cut into 3 mm fragments, and directly suspended in the extraction solution from the kit for cell lysis. The bead-beating time was extended to 20 min to yield sufficient quantities of DNA for SMRT sequencing, with reference to Albertsen et al.^{57 (link)}. SMRT sequencing was conducted using a PacBio Sequel system (Pacific Biosciences) in two independent runs according to the manufacturer’s standard protocols. SMRT libraries for CCS were prepared with a 4 kbp insertion length and two SMRT cells were used for each sample. Briefly, 3–5 kbp DNA fragments from each genomic DNA sample were extracted using the BluePippin size-selection system (Sage Science). Two sequencing libraries for CCS analysis were prepared using the SMRTbell Template Prep Kit 1.0-SPv3 according to the manufacturer’s protocol (Pacific Biosciences). The final libraries were sequenced using a PacBio Sequel sequencer with Sequel SMRT Cell 1M v2 and Sequel Binding/Sequencing Kits 2.0.

Bacterial community diversity was analysed via single‐molecule real‐time PacBio sequencing (Pacific Biosciences, Menlo Park, CA).³⁴, ³⁵ The full‐length 16S ribosomal RNA gene was amplified from genomic DNA using the bacteria‐specific primers 27 F (5′‐AGAGTTTGATCMTGGCTCAG) and 1492 R (5′‐TACGGYTACCTTGTTACGACTT). The quality and concentration of the PCR products were determined via electrophoresis on 2% agarose gels. Sequencing libraries were constructed using the SMRTbell Template Prep Kit 1.0‐SPv3 according to the manufacturer's instructions (Pacific Biosciences).
No sequences contained ambiguous bases. Details of the sequencing output, such as raw reads, clean reads and average read lengths, are summarized in supplemental Table S1. Operational taxonomic unit (OTU) clustering was performed using a 99% similarity cut‐off to represent one species per OTU. After sequencing data were rarefied, the alpha and beta diversities were calculated, and differences in relative abundance were compared using Statistical Analyses of Metagenomic Profiles (STAMP) software.

After bead purification with Agencourt AMPure XP (Beckman Coulter, USA), sequencing libraries were built using the SMRTbell Template Prep Kit 1.0‐SPv3 following the guidelines in the amplicon template protocol (Pacific Biosciences, USA). DNA damage repair, end-repair and ligation of hairpin adapters were performed according to the manufacturer’s instruction. DNA template libraries were bound to the Sequel polymerase 2.0 using the Sequel Binding Kit 2.0 (Pacific Biosciences, USA). The data collection per sample was done in a single Sequel SMRT Cell 1M v2 with 600 min movie time on the Sequel system (Pacific Biosciences, USA). We used a 5 pM on-plate loading concentration using Diffusion Loading mode and the Sequel Sequencing Plate 2.0 (Pacific Biosciences, USA).

The sequencing library of 1 μg total RNA from the mixed sample of C was performed using the SMRTbell™ Template Prep Kit 1.0-SPv3 (Pacific Biosciences, Menlo Park, CA, USA). The amount and concentration of the final library was verified with a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The size and purity of the library was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Following the Sequel Binding Kit 2.0 (Pacific Bioscience, USA) instruction for primer annealing and polymerase binding, the magbead-loaded SMRTbell template was performed on a PacBio Sequel instrument at Shanghai Oe Biotech Co., Ltd. (Shanghai, China).

Fresh leaf tissues from single-living black pepper plants were collected to extract genomic DNA and RNA (Supplementary Note 1). For genome survey analysis, a short paired-end Illumina DNA library with a 350 bp insert size (137 × coverage) was sequenced on the Illumina HiSeq 2500 sequencer. For PacBio Sequel sequencing, 50 µg of high-molecular-weight (HMW) genomic DNA were prepared to generate five standard SMRTbell libraries with 20 Kb insertions. PacBio long reads were sequenced using 15 SMRTcells on the PacBio Sequel System (Pacific Biosciences) with SMRTbell Template Prep Kit 1.0-SPv3 (Pacific Biosciences). HMW genomic DNA was also prepared for 10 × Genomics libraries according to the manufacturer’s protocol (Chromium Genome v1, PN-120229). Sequencing-read libraries were sequenced using HiSeq 2500 with 2 × 150 paired-end reads to generate ~96 Gb (120 × coverage) raw data.