Pacbio rs 2 sequencing platform

Manufactured by Pacific Biosciences

Sourced in United States

The PacBio RS II is a DNA sequencing platform developed by Pacific Biosciences. It utilizes single-molecule, real-time (SMRT) technology to sequence long DNA or RNA molecules. The core function of the PacBio RS II is to generate long read lengths and high-quality sequence data.

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Lab products found in correlation

Dneasy blood and tissue kit, by Qiagen (3 mentions) G tube, by Covaris (3 mentions) Trypticase soy broth (tsb), by BD (2 mentions) Agilent 2100 bioanalyzer high sensitivity kit, by Agilent Technologies (2 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions) Smrtbell template prep kit, by Pacific Biosciences (2 mentions) Pacbio smrtbell library preparation kit, by Pacific Biosciences (1 mentions) Pacbio rs 2 system, by Pacific Biosciences (1 mentions) Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions) Dna extraction kit, by Sparkjade (1 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Genomic tip 20 kit, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions) G tubes protocol, by Covaris (1 mentions) Gdna purification kit, by Qiagen (1 mentions) 454 gs flx genome sequencer, by Roche (1 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Miseq platform, by Illumina (1 mentions) Smrt portal, by Pacific Biosciences (1 mentions)

Close spelling variants of this product

PacBio RS II (187 citations) PacBio RS II platform (99 citations) RS II (40 citations) PacBio RS (13 citations) RS II sequencing platform (11 citations) PacBio sequencing (11 citations) PacBio sequencing platform (10 citations) PacBio platform (9 citations) Sequencing platform (4 citations) PacBio RS platform (3 citations)

24 protocols using pacbio rs 2 sequencing platform

Long-Read Genome Sequencing of Cyanobacteria

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The C. symbiosum cells were collected when the optical density (OD₆₀₀) reached 1.0 by centrifugation (10,000 × g for 10 min) and then immediately frozen in liquid nitrogen. Next, genomic DNA was extracted by using a DNA extraction kit (Shandong Sparkjade Biotechnology Co., Ltd., China).
Genome sequencing was carried out using the Pacific Biosciences (PacBio) RS II sequencing platform. In brief, genomic DNA was sheared into ∼10 kb fragments using a Covaris g-TUBE shearing device (Covaris, Woburn, Massachusetts, USA). DNA fragments were purified, end-repaired, and ligated to SMRTParkbell hairpin adapters using the PacBio SMRTbell library preparation kit (Pacific Biosciences, Menlo Park, China, USA). SMRTbell DNA libraries were constructed according to the manufacturer's protocol (Pacific Biosciences, Menlo Park, CA, USA). The library quality analysis and quantification were determined using the Qubit 2.0 Fluorometer (Bio-Medlab, China) and the Agilent 2100 Bioanalyzer system (Agilent Technologies, SantaClara, CA, USA). The SMRT sequencing was accomplished using the PacBioRSII system (Pacific Biosciences, Menlo Park, CA, USA) according to the standard protocol. The obtained continuous PacBio long-reads generated from SMRT sequencing runs were adopted for de novo assembly using the program Spades (version 3.14.1), yielding one circular chromosome.

Yang P., Tian J., Zhang L., Zhang H., Yang G., Ren Y., Fang J., Gu Y, & Jiang W. (2023). A toolbox for genetic manipulation in intestinal Clostridium symbiosum. Synthetic and Systems Biotechnology, 9(1), 43-54.

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PacBio Genome Sequencing of Altamira Strain

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Cells of the A. altamirensis strain C2P003 were cultured in R2 broth for two days at 25 °C and washed two times with 0.3% NaCl. Genomic DNA was extracted by the Genomic-Tip 20 Kit (Qiagen, Hilden, Germany) as described previously [19 (link)]. The fragment size, quantity and quality of DNA were assessed on a 1% agarose gel and with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). DNA was sequenced using the Pacific Biosciences (PacBio) RS II sequencing platform at Eurofins Genomics (Konstanz, Germany). Sequence reads were de novo assembled using the PacBio hierarchical genome assembly process (HGAP4). The assembling resulted in one contig with an average genome coverage of 124×. The genome sequence was circulated with the Circlator v. 1.5.5 [91 (link)].

Becker R., Ulrich K., Behrendt U., Schneck V, & Ulrich A. (2022). Genomic Characterization of Aureimonas altamirensis C2P003—A Specific Member of the Microbiome of Fraxinus excelsior Trees Tolerant to Ash Dieback. Plants, 11(24), 3487.

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PacBio Sequencing of S. coelicolor Genome

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Genomic DNA was purified from cultured S. coelicolor A3(2) cells using the genomic DNA (gDNA) purification kit (Qiagen). The genome was sequenced using the Pacific Biosciences (PacBio) RSII sequencing platform as previously described (28 (link),29 (link)). Briefly, SMRT bell libraries were constructed from a gDNA sample sheared to ∼10–20 kb using the G-tubes protocol (Covaris). The sheared ends were repaired and ligated to PacBio hairpin adapters according to the manufacturer's protocols. Incompletely formed SMRT bell templates and linear DNA were digested by Exonucleases III and VII (NEB). DNA quantification and the quality of the library was analyzed using the Qubit fluorimeter (Invitrogen, Eugene, OR) and 2100 Bioanalyzer (Agilent Technology). Two 18-kb SMRT bell libraries were prepared according to PacBio sample preparation protocols for 20 kb libraries and sequenced using C2-P4 chemistry (5 SMRT cells, 120 min collection times). Software provided by Pacific Biosciences or developed in house was used to detect modified bases (Interpulse duration, IPD ratios) in DNA sequencing. An IPD ratio >1 means that the sequencing polymerase slowed down (relative to the control) at this base position.

Lutz T., Flodman K., Copelas A., Czapinska H., Mabuchi M., Fomenkov A., He X., Bochtler M, & Xu S.Y. (2019). A protein architecture guided screen for modification dependent restriction endonucleases. Nucleic Acids Research, 47(18), 9761-9776.

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Hybrid Genome Sequencing Protocol

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PCR and Sanger sequencing was performed as previously described (Miller et al. 2014a (link)). Shotgun and paired-end (8–12 kb) 454 reads were obtained on a Roche 454 GS-FLX+ Genome Sequencer with Titanium chemistry using standard protocols. Illumina HiSeq reads were obtained from SeqWright (Houston, TX). SMRT sequencing was performed on the Pacific Biosciences (PacBio) RSII sequencing platform using 10 kb or 20 kb SMRTbell libraries, C2/C2 sequencing chemistry, and the 90-min data collection protocols. The SMRTbell libraries were prepared from 5 to 10 μg of bacterial genomic DNA, using the standard protocol from Pacific Biosciences (Menlo Park, CA, USA), and processed for sequencing as recommended by the supplier. A FASTQ file was generated from the PacBio reads using SMRTanalysis (ver. 2.1), and the reads were error-corrected using pacBioToCA with self-correction (Koren et al. 2013 (link)). The longest 20× of the corrected reads were assembled with Celera Assembler 8.1 (Koren et al. 2012 (link)). The resulting contigs were polished using Quiver (Chin et al. 2013 (link)).

Miller W.G., Yee E., Chapman M.H, & Bono J.L. (2017). Comparative Genomics of All Three Campylobacter sputorum Biovars and a Novel Cattle-Associated C. sputorum Clade. Genome Biology and Evolution, 9(6), 1513-1518.

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PacBio Whole Genome Sequencing of CFSAN061771

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The genomic DNA of strain CFSAN061771 was isolated from overnight culture using the DNeasy Blood and Tissue kit (Qiagen Inc., Valencia, CA) and sequenced on the Pacific Biosciences (PacBio) RS II sequencing platform as previously described^{46 (link)}. The sequences were annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP)^{47 (link)}.

Hammad A.M., Gonzalez-Escalona N., El Tahan A., Abbas N.H., Koenig S.S., Allué-Guardia A., Eppinger M, & Hoffmann M. (2022). Pathogenome comparison and global phylogeny of Escherichia coli ST1485 strains. Scientific Reports, 12, 18495.

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Mangrove-derived Bacterial Genome Sequencing

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A detailed description of sampling, isolation, and purification of the strains is available in [20 (link)]. Strains Bac330, Bac324 and Bac332 were isolated from mangrove mud samples collected from the Rabigh Harbor Lagoon in Saudi Arabia (39°0′35.762′′E, 22°45′5.582′′ N). Genome sequencing was performed at the Core Laboratory sequencing facility at KAUST using the PacBio RS II sequencing platform (Pacific Biosciences, USA) and assembled using PacBio’s SMRT Analysis pipeline v2.3.0. using default parameters. Prodigal [26 (link)] was used as the gene prediction method and genome annotation was completed using the Automatic Annotation of Microbial Genomes pipeline (AAMG) [27 (link)] with default parameters. A detailed description of sequencing, assembly, and annotation are available in [25 (link)].

Othoum G., Bougouffa S., Bokhari A., Lafi F.F., Gojobori T., Hirt H., Mijakovic I., Bajic V.B, & Essack M. (2019). Mining biosynthetic gene clusters in Virgibacillus genomes. BMC Genomics, 20, 696.

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Genomic DNA Extraction and Sequencing

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Freeze-dried mycelia were ground with liquid nitrogen and incubated in CTAB containing 1% β-Mercaptoethanol at 65 °C for 1 h. The supernatant was then extracted with an equal volume of chloroform-isoamyl alcohol (24:1). The extraction was repeated until no more precipitate formed. DNA was precipitated with 2:3 (vol:vol) of cold isopropanol and 1:10 (vol:vol) of 3 M NaAc (pH = 5.2), centrifuged at 10,000 rpm for 15 min at 4 °C, washed twice with 70% cold ethanol and treated with 1 ml of 10 mM Tris-HCl containing 20 μl of 100 mg/ml RNase A for 1 h at 37 °C. After chloroform-isoamyl alcohol extraction, and re-precipitation with cold isopropanol and NaAc at −20 °C for 2 h, DNA was washed twice, first with 70% and then with 100% cold ethanol. Air dried DNA was dissolved in 10 mM Tris-HCl (pH = 8.0). The amount and quality of total DNA was visualized by running out on a 1% agarose gel and quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific). A 20 K library was prepared with the total genomic DNA and three SMRT cells were sequenced using PacBio RS II sequencing platform (Pacific Biosciences, Nextomics Biosciences Co., Ltd., Wuhan).

Li Y., Hu X.D., Yang R.H., Hsiang T., Wang K., Liang D.Q., Liang F., Cao D.M., Zhou F., Wen G, & Yao Y.J. (2015). Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis. Scientific Reports, 5, 13892.

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Long-read sequencing of bacterial genomes

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For DH14, genomic DNA was extracted as described in [11 (link)], while for RACE1 the protocol described in [65 ] was used. Subsequently, SMRTbell™ genomic libraries were generated and sequenced at the Earlham Institute (formally known as The Genome Analysis Centre, Norwich, United Kingdom) and at the Max Planck Genome Centre in Cologne (Germany) for DH14 and RACE1, respectively. The Pacific Biosciences (PacBio) RSII sequencing platform with either P5C3 (DH14) or P6C4 (RACE1) chemistry was deployed (Pacific Biosciences, Menlo Park, CA; [66 (link)]). A total of 21 SMRT cells achieved ~ 50× coverage for RACE1 (1,115,202 reads, 8357 bp average size), while for DH14 6 SMRT cells resulted in ~ 25× coverage (1,478,871 reads, 4540 bp average size). In addition, DH14 genomic DNA was sequenced at ~ 50× coverage with the Illumina MiSeq platform, providing 2 × 300 bp paired-end reads.

Frantzeskakis L., Kracher B., Kusch S., Yoshikawa-Maekawa M., Bauer S., Pedersen C., Spanu P.D., Maekawa T., Schulze-Lefert P, & Panstruga R. (2018). Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen. BMC Genomics, 19, 381.

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High-Molecular Weight DNA Sequencing

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A single SMRT-bell library with 20 kb insert size was constructed from 10 μg of pure high-molecular weight DNA from one S. viminalis female (accession 78183) according to the manufacturer’s protocol (Pacific Biosciences). This library was sequenced on 48 SMRT cells using P5-C3 chemistry, and 4-h movies were captured for each SMRT cell using the PacBio RSII sequencing platform (Pacific Biosciences). Primary analysis and error correction of the raw data were done using SMRT Portal (Pacific Biosciences). After filtering, the mean read length was 8924 bp (longest read was 61 kbp) and a total of ~ 19.2 Gbp of data were recovered.

Almeida P., Proux-Wera E., Churcher A., Soler L., Dainat J., Pucholt P., Nordlund J., Martin T., Rönnberg-Wästljung A.C., Nystedt B., Berlin S, & Mank J.E. (2020). Genome assembly of the basket willow, Salix viminalis, reveals earliest stages of sex chromosome expansion. BMC Biology, 18, 78.

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High-Molecular-Weight DNA Extraction and PacBio Sequencing of L. monocytogenes

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High molecular weight DNA was extracted from L. monocytogenes cultures using Qiagen Genomic-tip 100/G columns and a modified manufacturer’s protocol as previously described [41 (link)] with the addition of mutanolysin with the proteinase K step followed by incubation at 50 °C for 1 h. Ten micrograms of DNA were sheared to a targeted size of 20 kb using a g-TUBE (Corvaris, Woburn, MA) and concentrated using 0.45X volume of AMPure PB magnetic beads (Pacific Biosciences, Menlo Park, CA) following the manufacturer’s protocol. Sequencing libraries were created using 5 μg of sheared, concentrated DNA and the PacBio DNA Template Prep Kit 2.0 (3Kb - 10Kb) according to the manufacturer’s protocol. The library was bound with polymerase P5 followed by sequencing on a Pacific BioSciences (PacBio) RS II sequencing platform with chemistry C3 and the 120 min data collection protocol.

Whitman K.J., Bono J.L., Clawson M.L., Loy J.D., Bosilevac J.M., Arthur T.M, & Ondrak J.D. (2020). Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers. BMC Veterinary Research, 16, 70.

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