Total DNA was extracted from muscle tissues of one male fish using a QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. Single-molecule real-time circular consensus sequencing (CCS) library preparation was conducted following the recommended protocols by Pacific Bioscience. In brief, a total of 50 μg genomic DNA was sheared to ~20 kb targeted size by using Covaris g-TUBEs (Covaris). The sheared genomic DNA was examined by Agilent 2100 Bioanalyzer DNA12000 Chip (Agilent Technologies) for size distribution. Sequencing libraries were constructed using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for HiFi sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer’s instructions. The constructed libraries were sequenced on one SMRT cell.
Pacbio rs 2 machine
Manufactured by Pacific Biosciences
Sourced in United States
The PacBio RS II is a single-molecule, real-time (SMRT) sequencing platform developed by Pacific Biosciences. It is designed to generate long, high-quality DNA sequences by detecting the incorporation of fluorescently labeled nucleotides during DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
G tube, by Covaris (4 mentions)
Pacbio dna template preparation kit 2, by Pacific Biosciences (4 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer dna12000 chip, by Agilent Technologies (2 mentions)
Bluepippin preparation system, by Sage Science (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Dna12000 chip, by Agilent Technologies (1 mentions)
Agencourt ampure beads, by Beckman Coulter (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nextera xt kit, by Illumina (1 mentions)
6 protocols using pacbio rs 2 machine
1
Single-Molecule Sequencing of Fish Genome
2
Genome Sequencing and Annotation Protocol
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Genomic DNA was sequenced using the Single Molecule Real-Time (SMRT) sequencing method with a PacBio RS II machine (Pacific BioSciences). The sequencing reads were assembled using the RS_ HGAP_ Assembly.3 workflow from the SMRT Portal 2.3. The assembled genome was annotated with the Rapid Annotation using Sub system Technology (RAST) using the RASTtk workflow (http://rast. nmpdr.org) (Overbeek et al. 2014) (link). Annotated protein sequences were further analyzed for conserved domains using Batch CD-Search (www. ncbi. nlm. nih. gov/ Structure/ bwrpsb/ bwrpsb. cgi) (Marchler-Bauer et al. 2015) (link). Multi-genome comparisons and identification of homologous virulence factor were performed with the Pathosystems Resource Integration Center (PATRIC) (Wattam et al. 2014) (link).
3
PacBio Sequencing of Verticillium dahliae
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The PacBio libraries were constructed using approximately 10 µg of genomic V. dahliae DNA that was mechanically sheared to a size of ~22 kb, using g-TUBES (Covaris, Inc., Woburn, MA) according the manufacturer’s instructions. PacBio SMRTbell libraries were prepared by ligation of hairpin adaptors at both ends of the DNA fragment (52 (link)) using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for SMRT sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer’s instructions. Libraries were purified using Agencourt AMPure beads (Beckman Coulter, Inc., Brea, CA) to remove short inserts of <1.5 kb. Libraries were size selected using the BluePippin preparation system (Sage Science, Beverly, MA) with a minimum cutoff of 7 kb. The sheared DNA and final library were characterized for size distribution using an Agilent Bioanalyzer 2100 (Agilent Technology, Inc., Santa Clara, CA) along with a DNA12000 chip (Agilent Technology, Inc.).
4
Muscle Tissue DNA Extraction and HiFi CCS Sequencing
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Total DNA was extracted from muscle tissue of one male fish using a QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions. Large-insert single-molecule real-time circular consensus sequencing (HiFi CCS) library preparation was conducted following the Pacific Biosciences recommended protocols. In brief, a total of 50 μg genomic DNA was sheared to ~20 kb targeted size by using Covaris g-TUBEs (Covaris). The sheared genomic DNA was examined by Agilent 2100 Bioanalyzer DNA12000 Chip (Agilent Technologies) for size distribution. Sequencing libraries ,were constructed using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for HiFi sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer's instructions. The constructed libraries were sequenced on one SMRT cell on a PacBio RSII sequencer.
5
PacBio Sequencing of Plasmid DNA
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Ten micrograms of plasmid was extracted and purified by amplification of the positive colony selected in the previous step. The PacBio libraries were constructed using a plasmid that was mechanically sheared to a size of ~22 kb using Covaris g-TUBE (Covaris, Inc., Woburn, MA) according the manufacturer’s instructions. PacBio SMRTbell libraries were prepared by ligation of hairpin adapters at both ends of the DNA fragment using the PacBio DNA template preparation kit 2.0 for SMRT sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc., Menlo Park, CA). A bluepippin preparation system (SAGE Science, Beverly, MA) was used to enrich more than 7 kb fragments in the library. Then, the quality of the library was tested by the Agilent Bioanalyzer 2100 kit (Agilent Technology, Inc., Santa Clara, CA). Sequencing was performed on the PacBio RS II instrument according to the manufacturer’s recommendations.
6
Genome Sequencing of Microbial Isolates
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All isolates underwent WGS (Illumina MiSeq or NextSeq 500). A subset of the isolates was re-sequenced by long-read sequencing using the PacBio Single-Molecular-Real-Time (SMRT) or Nanopore technology to complete the genome. The Illumina library preparation and sequencing were carried out as previously described (31 (link)). Briefly, DNA sequencing libraries were prepared using the Nextera XT kit (Illumina MiSeq system (Illumina, Netherlands BV, Eindhoven, the Netherlands) according to the manufacturer’s introductions and sequenced either on a MiSeq instrument with 2 × 300 cycles or on a NextSeq instrument with 2 × 150 cycles. The SMRT-sequencing was carried out on a PacBio RSII machine (Pacific Biosciences, MenloPark, CA, USA), as described earlier (31 (link)). Sequencing using Nanopore technology with a MinION sequencer was performed as described previously (32 (link)).
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