Bluepippin system

DNA was extracted from overnight liquid LB cultures grown at 37°C with the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Johannesburg, South Africa). The UCMB5007 and UCMB5044 genomes were sequenced using paired-end library preparation and an Illumina HiSeq 2000 at Macrogen Inc. (Republic of Korea). SMRT sequencing was carried out on a PacBio RSII machine (1 SMRT cell per strain) at the FGCZ, Zurich, Switzerland. Size selection was performed using the BluePippin system (SAGE Science, United States) as described before (Omasits et al., 2017 (link)) and resulted in fragments with an average subread length of 8–10 kb. Two 2 × 300 bp Illumina paired end libraries were prepared per strain using the Nextera XT DNA kit and sequenced on a MiSeq at Agroscope (Wädenswil, Switzerland). The At1 genome was sequenced with PacBio RSII CCS technology at Inqaba Biotec Ltd. (Pretoria, South Africa). DNA quality was evaluated spectrophotometrically at 260 and 280 nm light absorbance with a NanoDrop 1000 spectrometer (Thermofisher, South Africa). All raw DNA and RNA reads generated for this project by Illumina and PacBio sequencers are available from NCBI SRA database records accessible through the BioProject Web-sites. BioProject accession numbers are indicated in Table 1.

High-quality HMW DNA libraries for Oxford Nanopore MinION were constructed and DNA size selection was performed using BluePippin system (Sage Science, Inc.). Library preparation was performed with 1–1.7 μg DNA using the Ligation Sequencing Kit SQK-LSK109 (ONT, Oxford Nanopore Technologies) following manufacturer’s guidelines. Libraries were loaded on MinION FLO-MIN106D flow cell. Base calling was done using the GPU version of Guppy v2.1. “Dulce” samples produced 1.7 million sequences with a sum length of 23.3 Gb between 70 bp and– 148 592 bp with an average length of 13 729 bp. “Tam Dew” produced 1.7 million sequences with a sum length of 15.7 Gb between 76 bp and 117 396 bp with an average length of 117 396 bp. Mean read qualities for both samples were equal or above Q10.

Samples (20 μg) of DNA (OD_260/OD₂₈₀ ≈ 1.8) were sheared using a Covaris® g-TUBE® device (Covaris, USA), diluted to 200–300 ng/μL in elution buffer, and centrifuged at 5,500 rpm (2029 g) for 2 min on a MiniSpin Plus (Eppendorf). We constructed SMRTbell libraries according to the Procedure & Checklist-20 kb Template Preparation using the BluePippin™ Size Selection protocol (http://files.pacb.com/Training/IntroductiontoSMRTbellTemplatePreparation/story_content/external_files/Introduction%20to%20SMRTbell%E2%84%A2%20Template%20Preparation.pdf). Briefly, the library was run on a BluePippin system (Sage Science, MA, USA) to select SMRTbell templates > 10 kb. Sequencing primers were annealed to the hairpins of the templates and bound with P5 sequencing polymerase and MagBeads (Pacific Biosciences, CA, USA). The libraries were sequenced on a PacBio RS II platform at Shanghai Personal Biotechnology Cp. Ltd.

For DNA extraction, fresh leaves of H. citrina (cultivar Datong hua) were collected from Datong, Shanxi Province, China. High-quality genomic DNA was isolated employing a modified CTAB protocol (Porebski et al., 1997 (link)). The purity, concentration, and integrity of the extracted DNA were examined using a NanoDrop (Thermo Scientific, USA), a Qubit fluorometer (Thermo Scientific, USA), and 0.35% agarose gel electrophoresis, respectively. Both short-read (Illumina) and long-read (Oxford Nanopore) sequencing technologies were implemented to obtain full-length mitogenome sequences. To collect large DNA sequences, optional fragmentation was performed using the BluePippin system (Sage Science, USA). Subsequently, the fragmented DNA was treated using damage repair, end preparation, A-tailing, and adapter ligation and then cleaned with magnetic beads. A library > 8 kb was constructed following the SQK-LSK109 (Oxford Nanopore Technologies, ONT, UK) sequencing kit protocol and loaded into a Nanopore GridION Sequencer (ONT, UK).

The cDNAs were prepared using the SMARTer PCR cDNA Synthesis Kit (Clontech) from 1 μg of purified polyA(+) RNA. cDNAs were then size selected (0.5–1, 1–2, 2–3, and 3–6 kb) using the BluePippin™ system (Sage Science). Then, Iso‐seq libraries were constructed according to the supplied protocol (http://www.pacb.com/wp-content/uploads/2015/09/Procedure-Checklist-Isoform-Sequencing-Iso-Seq-using-the-Clontech-SMARTer-PCR-cDNA-Synthesis-Kit-and-the-BluePippin-Size-Selection-System.pdf). For each of the four fraction size libraries, three single‐molecule real‐time (SMRT) cells were sequenced using P6‐C4 reagent on the PacBio RS II platform with 4 h sequencing movies for the leaf and grain tissues, respectively.