Smrtbell express template prep kit 2

The DNA was extracted from the colonic content samples using a QIAamp DNA Isolation Kit (Qiagen, Hilden, Germany). Nanodrop 2000 was used to check the quantity and quality of the extracted DNA. The full-length bacterial 16S rRNA gene was amplified with primer pairs: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) by an ABI GeneAmp 9700 PCR thermocycler (ABI, Los Angeles, CA, USA). Purified amplicons were pooled in an equimolar and a DNA library was constructed using the SMRTbell Express Template Prep Kit 2.0 (PacBio, Menlo Park, CA, USA). Purified SMRTbell libraries were sequenced on a Pacbio Sequel II System (PacBio, Menlo Park, CA, USA) according to the standard protocols by Majorbio Bio-Pharm Technology (Shanghai, China). The raw 16S rRNA gene sequencing reads were demultiplexed to circular consensus sequence (CCS) reads by SMRTLink version 8.0 and length-filtered (<1000 or >1800 bp). Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE version 7.1; chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.11 against the 16S rRNA database (Silva v138) with a confidence threshold of 0.7. All obtained raw sequence datasets have been uploaded to the NCBI Sequence Read Archive (SRA) with the accession number PRJNA988420.

The quality of the extracted DNA was measured with a Genomic DNA ScreenTape System (Agilent) and a Qubit Fluorometer (Thermo Fisher Scientific). A long-read DNA library was prepared with the SMRTbell Express Template Prep Kit 2.0 (PacBio) and sequenced using the PacBio Sequel lle system in CCS mode (PacBio). The resulting raw data were converted to FASTQ format using BAM2fastx 1.3.1 (PacBio). Reads longer than 5 kb were extracted with SeqKit 0.15.0 (Shen et al. 2016 (link)) and used for genome assembly with the Hifiasm 0.15.5-r350 assembler (Cheng et al. 2021 (link)).The -l 0 option was specified to disable the purge haplotigs function since the plant was monohaploid.

HiFi SMRTbell libraries were constructed according to the manufacturer’s manual by using SMRTbell Express Template Prep Kit 2.0 (PacBio, 100-938-900). For all samples, ~500Rng of cDNA obtained by performing LAP-CAP from the previous step was used for library preparation. The library construction includes DNA damage repair (37 °C for 30 min), end-repair/A-tailing (20 °C for 30 min and 65 °C for 30 min), adaptor ligation (20 °C for 60 min) and purification with 0.6× SPRIselect beads.

High Molecular Weight (HMW) gDNA was extracted using Circulomics NanoBind Tissue Big DNA
kit (PacBio, CA, USA). gDNA was sheared to ∼10–15 kb fragments using Covaris g-Tube
(Covaris, LLC). For long-read sequencing, the Pacbio SMRTbell Library was prepared
according to the manufacturer's protocol using the SMRTbell express template prep kit 2.0
(PacBio). Quality control was performed using Qubit HS dsDNA assay kit and TapeStation for
library integrity check. Sequencing primer annealing and Polymerase Binding were carried
out using the Sequel II binding kit 2.0, Internal Control 1.0, and Sequencing Primer v4.
The bound complex was sequenced on a Sequell II system running SMRTcell 8M. For short read
sequencing, fragment lengths were verified using the Bioanalyzer (Agilent Technologies,
Palo Alto, CA, USA). The sequencing library was prepared using a MisSeq library prep kit
according to the manufacturer's instructions. Library fragments were sequenced on the
Illumina NextSeq sequencing platform.