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Pacbio rs 2 single molecule real time

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The PacBio RS II is a Single Molecule Real Time (SMRT) sequencing platform that enables long-read sequencing. It utilizes zero-mode waveguides to detect single DNA molecules in real-time, generating long sequence reads with high accuracy.

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Lab products found in correlation

Wizard genomic dna purification kit, by Promega (7 mentions) Tbs 380 fluorometer, by Promega (3 mentions) Novaseq600, by Illumina (1 mentions) Hiseq x ten machine, by Illumina (1 mentions) Nextflex rapid dna seq kit, by Illumina (1 mentions) Nebnext ultra dna library prep kit for, by Illumina (1 mentions) Clc genomics workbench, by Illumina (1 mentions) Nextseq 500, by Illumina (1 mentions)

11 protocols using pacbio rs 2 single molecule real time

1

Genome Analysis of Tigecycline-Resistant Bacteria

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To identify the genomic alterations of K2606 after exposure to tigecycline, we sequenced the whole genomes of K2606 and its two derivatives (K2606-4 and K2606-16). Genomic DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega) according to the manufacture’s protocol. Genomes were sequenced using a combination of PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platforms, and the resulting data were used for bioinformatics analysis. Glimmer was used for CDS prediction, tRNA-scan-SE was used for tRNA prediction and Barrnap was used for rRNA prediction. The predicted CDSs were annotated from NR, Swiss-Prot, Pfam, GO, COG and KEGG database using sequence alignment tools such as BLAST, Diamond and HMMER. Briefly, each set of query proteins was aligned with the databases, and annotations of best-matched subjects (e-value <10−5) were obtained for gene annotation.
Genome of K2606 was used as a reference. Variants (SNPs, short indels, and large deletions) of K2606-4 and K2606-16 were called based on the Bowtie2 assembled data using GATK toolkit by Broad Institute. The variants were verified by universal PCR and sequencing.
Li J., Xu Q., Ogurek S., Li Z., Wang P., Xie Q., Sheng Z, & Wang M. (2020). Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin–Tazobactam and Ceftolozane–Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae. Infection and Drug Resistance, 13, 4309-4319.
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2

Genomic DNA Isolation and Sequencing of B. halotolerans

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High-quality genomic DNA isolated from B. halotolerans LDFZ001 with Wizard® Genomic DNA Purification Kit (Promega) according to manufacturer’s protocol was quantified with TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA), and applied to a combination of PacBio RS II Single MoleculeReal Time (SMRT) and Illumina sequencing platforms for sequencing. Then, data generated were analyzed using I-Sanger Cloud Platform (www.i-sanger.com) from Shanghai Majorbio (Shanghai, China).
Feng Z., Xu M., Yang J., Zhang R., Geng Z., Mao T., Sheng Y., Wang L., Zhang J, & Zhang H. (2022). Molecular characterization of a novel strain of Bacillus halotolerans protecting wheat from sheath blight disease caused by Rhizoctonia solani Kühn. Frontiers in Plant Science, 13, 1019512.
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3

Complete Genome Sequencing of Strain JP233

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For DNA extraction, the stored JP233 culture was streaked onto LB plate and incubated in a growth chamber at 28 °C for 3 days. A single pure colony was inoculated into a sterile tube containing 5 mL LB broth, which was then grown in an orbital shaker (180 rpm) for 48 h at 28 °C. Strain JP233 was harvested and genomic DNA was extracted using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA), following the manufacturer’s instructions.
The JP233 genome was sequenced using a combination of PacBio RS II Single Molecule Real Time (SMRT) and Illumina NovaSeq 600 sequencing platforms by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). For Illumina sequencing, DNA samples were sheared into 400 bp–500 bp fragments and used to build the sequencing library. The prepared library was then used for paired-end Illumina sequencing using the PE150 strategy. For PacBio sequencing, a 10 kb insert library was prepared and sequenced on one SMRT cell using standard methods.
Wang L., Zhou F., Zhou J., Harvey P.R., Yu H., Zhang G, & Zhang X. (2022). Genomic Analysis of Pseudomonas asiatica JP233: An Efficient Phosphate-Solubilizing Bacterium. Genes, 13(12), 2290.
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4

Genome Sequencing of Lactobacillus plantarum JS21

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First, L. plantarum JS21 culture was subcultured twice in MRS broth followed by incubation anaerobically at 37 °C for 24 h. A 50-mL fresh culture was pipetted onto a sterile tube and centrifuged at 6000× g for 10 min at 4 °C. Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Purified genomic DNA was quantified by TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA, USA). High quality DNA (OD260/280 = 1.8~2.0, >20 μg) was used to carry out further research. Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) sequenced the whole genome of L. plantarum JS21 using two different sequencing techniques. The genome was sequenced using a combination of PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platforms. The Illumina data were used to evaluate the complexity of the genome.
Liu Y., Wang S., Wang L., Lu H., Zhang T, & Zeng W. (2024). Characterization of Genomic, Physiological, and Probiotic Features of Lactiplantibacillus plantarum JS21 Strain Isolated from Traditional Fermented Jiangshui. Foods, 13(7), 1082.
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5

Genome sequencing of Streptomyces lydicus M01

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Streptomyces lydicus M01 were cultured in the ISP-2 medium at 28°C for 48 h. The genomic DNA extraction was performed using Wizard® Genomic DNA Purification Kit (Promega, Madison, MI, United States) according to manufacturer’s protocol. The complete genome sequencing and assembly were performed by the Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China. Genomic DNA was sequenced using a combination of PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platforms. The data generated from PacBio and Illumina platform were used for bioinformatics analysis using the online platform of Majorbio Cloud Platform.1 Glimmer (Delcher et al., 2007 (link)) was used for CDS prediction, tRNA-scan-SE (Borodovsky and McIninch, 1993 ) was used for tRNA prediction, and Barrnap was used for rRNA prediction. The predicted CDSs were annotated from NR, Swiss-Prot, Pfam, GO, COG, and KEGG databases. The biosynthetic gene clusters of secondary metabolites of S. lydicus M01 were predicted using the online antiSMASH v6.0.1 software (Blin et al., 2021 (link)).
Wang M., Li J., Cong W, & Zhang J. (2022). Antimicrobial Mechanism and Secondary Metabolite Profiles of Biocontrol Agent Streptomyces lydicus M01 Based on Ultra-High-Performance Liquid Chromatography Connected to a Quadrupole Time-of-Flight Mass Spectrometer Analysis and Genome Sequencing. Frontiers in Microbiology, 13, 908879.
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6

Whole-genome Sequencing of E. faecalis

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Whole-genome sequence analysis of Enterococcus faecalis strain ATCC19433 was conducted utilizing the PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platforms, carried out by Majorbio Co. in Shanghai, China. A PacBio library with an average insert size of ~ 10 kb was constructed, followed by high-throughput sequencing.
Zheng Y., Duan Z., Wu Y., Luo Y., Peng X, & Wu J. (2024). Analysis of the Cadmium Removal Mechanism of Human Gut Bacteria Enterococcus faecalis Strain ATCC19433 from a Genomic Perspective. Biological trace element research.
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7

Whole-Genome Sequencing of Salmonella Typhimurium

Cited 1 time
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The whole S. Typhimurium SH160 genome was sequenced using PacBio RS II Single Molecule Real-Time (SMRT) and Illumina sequencing platforms. The raw reads were then assembled into a contig using the hierarchical genome assembly process (HGAP) and Canu.10 (link),11 (link) The complete plasmid sequence was predicted with GLIMMER and annotated with the NR, Swiss-Prot, Pfam, GO, COG, and KEGG databases using sequence alignment tools, including BLAST, Diamond and HMMER.12 (link)–15 (link) Easyfig was used in comparative analysis (Genbank accession number KY435936, KF220657, KF877335).16 (link),17 (link) ResFinder and PlasmidFinder were used to detect drug-resistant genes and the plasmid sequencing type.18 (link),19 (link)
Gao Y., Wen J., Wang S., Xu X., Zhan Z., Chen Z., Bai J., Qu X., Zhang H., Zhang J, & Liao M. (2020). Plasmid-Encoded blaNDM-5 Gene That Confers High-Level Carbapenem Resistance in Salmonella Typhimurium of Pork Origin. Infection and Drug Resistance, 13, 1485-1490.
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8

Genomic DNA Extraction and Sequencing

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Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). The genome was sequenced using a combination of PacBio RS II Single MoleculeReal Time (SMRT, Singapore, Singapore) and Illumina sequencing platforms. Illumina sequencing libraries were prepared from the sheared fragments using the NEXTflex™ Rapid DNA-Seq Kit. The prepared libraries then were used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten machine.
Wang L., Qiao L., Li A., Chen L., He B., Liu G., Wang W, & Fang J. (2023). Integrative Multiomics Analysis of the Heat Stress Response of Enterococcus faecium. Biomolecules, 13(3), 437.
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9

Hybrid Plasmid Sequencing: Illumina and PacBio

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Plasmid sequencing was conducted on an Illumina platform and a PacBio RSII single-molecule real-time (SMRT) sequencing platform. The Illumina paired-end libraries were constructed with the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) and sequenced on an Illumina NextSeq 500 platform. SMRT sequencing was performed at Wuhan Institute of Biotechnology, China. De novo assemblies of PacBio RSII reads and Illumina reads were performed by the hierarchical genome assembly process (HGAP, Pacific Biosciences) and the CLC Genomics Workbench (CLC bio, Denmark) respectively. Long assembled contigs obtained from PacBio reads were used to align and join the contigs obtained from the Illumina assembly results. The completed plasmid sequence was confirmed by PCR and then annotated with the RAST tool [34 (link)] and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Each plasmid was sequenced using both Illumina and PacBio platforms and only high-quality data were used for further analysis.
Chen K., Chan E.W, & Chen S. (2019). Evolution and transmission of a conjugative plasmid encoding both ciprofloxacin and ceftriaxone resistance in Salmonella. Emerging Microbes & Infections, 8(1), 396-403.
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10

Whole-Genome Sequencing of Clinical Isolates

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Two isolates from patients with typical clinical presentation were selected for whole-genome sequencing. Draft genome sequencing was performed on the Ilumina HiSeq platform, while complete genomes were sequenced on PacBio RS II Single Molecule Real Time and Illumina sequencing platforms at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).
Yang Z., Qin J., Zhao L., Chen T., Huang Q., Jian Y., Zhao Q., Yang S., Li Q., Liu Q., Otto M, & Li M. (2023). Host sorbitol and bacterial sorbitol utilization promote C. difficile infection in inflammatory bowel disease. Gastroenterology, 164(7), 1189-1201.e13.
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