For each genomic DNA sample, a 10 kb SMRTbell library was constructed according to manufacturer’s instructions (PacBio, Menlo Park, CA, USA). Each sample was first sheared and treated with Exo VII, followed by the DNA damage repair and end-repair steps. Following end-repair, barcoded adapters are ligated to the sample. Samples were then pooled and underwent Exo III and VII treatments, followed by two 0.45X AMPure PB bead purification rounds. The libraries were sequenced using a PacBio RSII instrument and P6C4 chemistry (PacBio, CA, USA).
Rsii instrument
Manufactured by Pacific Biosciences
Sourced in United States
The RSII instrument is a high-performance, long-read sequencing system developed by Pacific Biosciences. It utilizes the company's proprietary Single Molecule, Real-Time (SMRT) technology to generate long, accurate DNA sequence reads. The RSII instrument enables researchers to explore complex genomic structures and gain insights into biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Hgap v3, by Pacific Biosciences (2 mentions)
Kod hot start master mix, by Merck Group (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Smrtbell library, by Pacific Biosciences (1 mentions)
P6 c4 chemistry, by Pacific Biosciences (1 mentions)
Bluepippin system, by Sage Science (1 mentions)
Dna polymerase binding kit p6 v2, by Pacific Biosciences (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Smrt analysis software v2, by Pacific Biosciences (1 mentions)
Genomic tip, by Qiagen (1 mentions)
Maxwell lev blood dna kit, by Promega (1 mentions)
Maxwell 16, by Promega (1 mentions)
Miseq benchtop sequencer, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Smrt analysis v2, by Pacific Biosciences (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Ultrasonication, by Covaris (1 mentions)
26 protocols using rsii instrument
1
PacBio SMRT Sequencing Library Prep
2
Detecting HBV Integration Sites via Long-Read Sequencing
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Whole-genome long sequence data were obtained using the single-molecule real-time (SMRT) sequencing method with the PacBio RSII instrument. Long-insert sequence libraries (SMRTbell-10kb) were prepared from 20 μg of paired tumor and non-cancerous tissue DNA from HX25, enriched to >4 kb using BluePippin, loaded on SMRT Cell v3, and sequenced with P4C2 chemistry according to the manufacturer’s instructions.
The sequence reads (tumor: 15.64 average depth, 1783 bp average mapped read length, and 85.35% mapped accuracy; non-cancerous: 11.36 average depth, 4082 bp average mapped read length, and 84.99% mapped accuracy) were mapped using BLASR41 (link). Long reads that included at least two HBV integration sites or included both an integration site and a breakpoint of structural rearrangement of HBV genome sequences were selected. Each integration site or breakpoint in the read was validated by the detection of integration or rearrangement of HBV using the WGS short reads described above.
The sequence reads (tumor: 15.64 average depth, 1783 bp average mapped read length, and 85.35% mapped accuracy; non-cancerous: 11.36 average depth, 4082 bp average mapped read length, and 84.99% mapped accuracy) were mapped using BLASR41 (link). Long reads that included at least two HBV integration sites or included both an integration site and a breakpoint of structural rearrangement of HBV genome sequences were selected. Each integration site or breakpoint in the read was validated by the detection of integration or rearrangement of HBV using the WGS short reads described above.
3
Genomic and Transcriptomic Data of Zymoseptoria Species
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Details regarding the individual Zymoseptoria isolates can be found in Table 1 . For genomic data we used the three Z. tritici isolates IPO323 (reference), Zt05 and Zt10, one Z. ardabiliae isolate (Za17), one Z. brevis isolate (Zb87), one Z. passerinii isolate (Zpa63), and the Z. pseudotritici isolate (Zp13). For transcriptomic and epigenomic data we used Z. tritici Zt09 (IPO323 ΔChr18) a derivate of the reference isolate IPO323 deleted with the chromosome 18 [31 (link)].
Long read assemblies of the Z. tritici isolates Zt05 and Zt10 were described and published previously [30 ]. For DNA extraction and long read sequencing cultures of Z. pseudotritici, Z. ardabiliae, Z. brevis and Z. passerinii were maintained in liquid YMS medium (4 g/L yeast extract, 4 g/L malt extract, 4 g/L sucrose) at 200 rpm and 18 °C. DNA extraction was conducted as previously described [26 (link)]. PacBio SMRTbell libraries were prepared using DNA extracted from single cells based on a CTAB extraction protocol [30 , 46 (link)]. The libraries were size selected with an 8-kb cutoff on a BluePippin system (Sage Science).
After selection, the average fragment length was 15 kb. Sequencing of the isolates Za17, Zb87, and Zp13 was run on a PacBio RS II instrument at the Functional Genomics Center, Zurich, Switzerland. Sequencing of the Zpa63 isolate was performed at the Max Planck-Genome-Centre, Cologne, Germany.
Long read assemblies of the Z. tritici isolates Zt05 and Zt10 were described and published previously [30 ]. For DNA extraction and long read sequencing cultures of Z. pseudotritici, Z. ardabiliae, Z. brevis and Z. passerinii were maintained in liquid YMS medium (4 g/L yeast extract, 4 g/L malt extract, 4 g/L sucrose) at 200 rpm and 18 °C. DNA extraction was conducted as previously described [26 (link)]. PacBio SMRTbell libraries were prepared using DNA extracted from single cells based on a CTAB extraction protocol [30 , 46 (link)]. The libraries were size selected with an 8-kb cutoff on a BluePippin system (Sage Science).
After selection, the average fragment length was 15 kb. Sequencing of the isolates Za17, Zb87, and Zp13 was run on a PacBio RS II instrument at the Functional Genomics Center, Zurich, Switzerland. Sequencing of the Zpa63 isolate was performed at the Max Planck-Genome-Centre, Cologne, Germany.
4
Long-Read DNA Sequencing Methods
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DNA sequence data were obtained primarily using the Pacific Biosciences (PacBio) Single Molecule, Real-Time (SMRT) system with DNA/Polymerase Binding Kit P6 v2, MagBead Kit v1 and DNA Sequencing Reagent Kit 4.0 v2 at the PacBio Sequencing Services of the University of Washington (Seattle, WA, USA). Long DNA reads were collected in one SMRT cell on a PacBio RSII instrument. Additional long reads were obtained using the Oxford Nanopore Technologies (ONT) MinION system. The MinION genomic DNA library was prepared according to the ONT Nanopore Sequencing Kit SQK-MAP006 protocol and quantified with Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, USA). Sequencing reads were generated on Flow Cells (Flo-MAP003) with MinKNOW 0.51.1.62 and base-called with Metrichor 2.39. "2D pass" reads were converted into FASTA format with poretools v. 0.5.1. These reads are the highest quality reads, selected from reads where both strands of a double-stranded fragment were analyzed together in one pore.
5
High-Quality E. coli Genome Sequencing
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Genomic DNA of E. coli 50579417 was prepared from an overnight culture using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The DNA library was prepared using the Pacific Biosciences 20-kb library preparation protocol and size selection with a 9-kb cutoff using BluePippin (Sage Sciences, Beverly, MA). Sequencing was performed using the Pacific Biosciences RSII instrument using P6-C4 chemistry with a 360-min movie time and one single-molecule real-time sequencing (SMRT) cell. The sequences were assembled and polished at The Norwegian Sequencing Centre (https://www.sequencing.uio.no/ ) using HGAP v3 (Pacific Biosciences, SMRT Analysis Software v.2.3.0). Minimus2 from AMOS was used to circularize contigs, and RS_Resequencing.1 software (Pacific Biosciences, SMRT Analysis Software v.2.3.0) was used for correction of bases after circularization.
6
High-Quality M. tuberculosis DNA Extraction
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M. tuberculosis H37Ra (ATCC25177) was obtained from ATCC and cultured on Lowenstein-Jenson slants and Middlebrook 7H11 plates. Cultures were incubated until growth of a full bacterial lawn. DNA was extracted using Genomic-tips (Qiagen Inc.) following the manufacturer’s sample preparation and lysis protocol for bacteria with the following modifications. Culture was harvested directly into buffer B1/RNAse solution, homogenized by vigorous vortex mixing and inactivated at 80°C for 15 minutes. Lysozyme was added and incubated at 37°C for 30 minutes followed by the addition of proteinase K and further incubation at 37°C for an additional 60 minutes. Buffer B2 was added and the mixture was incubated overnight at 50°C. Wide-bore pipet tips were used to optimize recovery of large DNA fragments. The remainder of the Genomic-tip protocol was carried out exactly as described by the manufacturer. DNA purity and concentration was analyzed on a Nanodrop 1000 (Thermo Scientific). The DNA was then sequenced using two SMRTCells on the Pacific Biosciences RS II instrument with the P6-C4 chemistry and a 20kb insert library preparation.
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M. tuberculosis H37Ra (ATCC25177) was obtained from ATCC and cultured on Lowenstein-Jenson slants and Middlebrook 7H11 plates. Cultures were incubated until growth of a full bacterial lawn. DNA was extracted using Genomic-tips (Qiagen Inc.) following the manufacturer’s sample preparation and lysis protocol for bacteria with the following modifications. Culture was harvested directly into buffer B1/RNAse solution, homogenized by vigorous vortex mixing and inactivated at 80°C for 15 minutes. Lysozyme was added and incubated at 37°C for 30 minutes followed by the addition of proteinase K and further incubation at 37°C for an additional 60 minutes. Buffer B2 was added and the mixture was incubated overnight at 50°C. Wide-bore pipet tips were used to optimize recovery of large DNA fragments. The remainder of the Genomic-tip protocol was carried out exactly as described by the manufacturer. DNA purity and concentration was analyzed on a Nanodrop 1000 (Thermo Scientific). The DNA was then sequenced using two SMRTCells on the Pacific Biosciences RS II instrument with the P6-C4 chemistry and a 20kb insert library preparation.
7
Whole Genome Sequencing of Propionibacterium freudenreichii
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Nineteen Propionibacterium freudenreichii samples were sequenced with the Pacific Biosciences RS II Instrument using either P4/C2 or P5/C3 chemistries (listed in Table 2 ). Two SMRT cells were used for each sample. Movie times varied from 120 to 240 min. The total number of obtained bases and subreads and their mean and N50 lengths are listed in Table 2 . The Hierarchical Genome Assembly Process (HGAP) V3 implemented in the SMRT Analysis package (v.2.3.0) was used to generate de novo genome assemblies with default parameters, excluding the genome size estimate parameter which was set to 3,000,000 bp. Obtained circular sequences were polished using SMRT Analysis RS Resequencing protocol and the Quiver consensus algorithm. Chromosomal genome sequences were set to begin from the chromosomal replication initiator protein (dnaA). The sequences were then annotated with Prodigal v. 2.6.2. All sequences were deposited in the European Nucleotide Archive (ENA). Complete genome, phage and plasmid sequence sizes, sequencing coverages, GC percentages, number of predicted genes and ENA accession numbers are listed in Table 1 . Base modifications and motifs were detected using RS Modification and Motif analysis protocol (SMRT Analysis package v.2.3.0).
8
Multidrug-Resistant Pathogen Genome Sequencing
9
Synthetic Mixture of E. coli Strains
10
Hybrid Genome Assembly of Izh-4 Isolate
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DNA of isolate Izh-4 was submitted to WGS using SMRT sequencing on the Pacific BioScience Technology platform. The sequencing service was provided by the core facility located at the Norwegian Sequencing Centre (NSC) (www.sequencing.uio.no ). DNA was extracted from 64 × 109 cells using a Maxwell® 16 and a Maxwell LEV Blood DNA kit (Promega, Germany). The 20 kb library preparation protocol was employed. Size selection of the final library was performed using 0.4x Amp beads. The library was sequenced on a Pacific Biosciences RS II instrument using P6-C4 chemistry with 360 min movie time, two SMRT cells were used for sequencing due to poor loading. De novo assembly was performed using hierarchical genome assembly process (HGAP v3, Pacific Biosciences, SMRT Analysis Software v2.3.0) with default parameters (expected genome size 1.6 Mb, minimum target coverage 15X). RS_Resequencing.1 software (SMRT Analysis version v2.3.0) was used to map SMRT reads back to sequences in order to correct contigs after assembly clean-up. PacBio contigs were polished by mapping Illumina pair-end reads using Pilon v1.22.
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