Pacbio smrtbell express template prep kit 2

High molecular weight DNA was sheared in a Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain an average fragment size of 10 kb. After shearing the DNA, size distribution was checked on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). Five hundred nanograms of the DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s recommendations. No size selection was applied. The pooled bar-coded libraries were sequenced with v3.0/v3.0 chemistry and diffusion loading on a PacBio Sequel instrument (Pacific Biosciences, Menlo Park, CA, USA) at 600 min movie length, pre-extension time of 120 min, using one SMRT cell 1 M v3.
Genome assembly was performed using CANU 2.0 with the option ‘pacbio-raw’ and a defined expected genome size of 5 Mbp^{72 (link)}. The circularization of the genomes was achieved using Circlator v.1.5.5 with default parameter settings^{73 (link)}. Genes were predicted using Prokka v.1.14.6^{74 (link)} but, during the submission of the sequenced genomes, reassigned through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP, version 4.11). Sequencing details and NCBI accession numbers are summarized in Supplementary Data file 4.

Library preparations and sequencing were performed at Lausanne Genomic essentially as recently described (39 (link)). Briefly, high-molecular weight DNA was sheared with Megaruptor (Diagenode, Denville, NJ, USA) to obtain 10- to 15-kb fragments. After shearing the DNA, size distribution was checked on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). A total of 500 ng of DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Express Template Prep kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s recommendations. The resulting library was pooled with other libraries which were processed in the same manner. The pool was size-selected with Ampure PacBio beads to eliminate fragments of <3kb. It was sequenced with v2.0/v2.0 chemistry and diffusion loading on a PacBio Sequel II instrument (Pacific Biosciences, Menlo Park, CA, USA) at a 900-min movie length and a pre-extension time of 120 min using one SMRT cell 8 M.

The yellow husk/pericarp mutant line yw1 was obtained from the following process: dry black‐coloured W1 seeds were treated with gamma rays at a dosage of 300 Gy. After the treatment, the seeds were continuously planted in the field (Hubei, China). A stably inherited mutant with yellow husk/pericarp trait was identified from the M₄ generation, which was designated as yw1. Genomic DNA was extracted from the yw1 fresh leaves using DNeasy Plant Mini Kit (QIAGEN). The SMRT bell library was constructed using the Pacific Biosciences (PacBio) SMRT bell express template prep kit 2.0. Sequencing was carried out on the PacBio Revio platform of Benagen Technology Co., Ltd. (Wuhan, China). The SMRTlink v11.0 software was used to clean up the raw PacBio read data. Minimap2 (v2.17) was used to align the long‐reads to the Hor13821 genome, which was further sorted by samtools v1.9. Previously published short‐gun sequencing reads (SRR3655669—SRR3655670) for wild type W1 were downloaded from the ENV database (https://www.ebi.ac.uk/ena/browser/; Tan et al., 2020 ) and were mapped to the Hor13821 genome using bwa‐mem2 (v2.2.1) tool. All mapped reads were visualized using the Samplot tool (https://www.github.com/ryanlayer/samplot; Belyeu et al., 2021 (link)).