Genomic DNA extraction, library preparation, and whole-genome sequencing on a NextSeq 500 platform (Illumina, Inc.), using 2 × 150-bp chemistry, was performed as previously described (24 (link)). One isolate (designated NZ-SC16875) underwent genomic DNA extraction with the GenElute bacterial genomic DNA kit (Sigma-Aldrich), followed by sequencing on the RSII (Pacific Biosciences) with P6-C4 chemistry according to the 20-kb template preparation using the BluePippin size selection system protocol (Pacific Biosciences). Sequences were initially analyzed using the Nullarbor pipeline (T. Seeman; https://github.com/tseemann/nullarbor ). Phage regions were detected using Phaster (25 (link)). Maximum likelihood trees were inferred using IQ-TREE (26 (link)). Recombination was detected using Gubbins (version 2.2.0-1) (27 (link)), and population structure was investigated using hierarchical Bayesian analysis with hierBAPS (28 (link)). Additional information on bioinformatic analyses is provided in the Supplemental Methods.
Bluepippin size selection system protocol
Manufactured by Pacific Biosciences
The BluePippin size selection system is a laboratory instrument designed for automated DNA size selection. It utilizes a patented technology to precisely separate DNA fragments based on their size, enabling users to isolate targeted DNA regions from complex samples. The BluePippin system provides a streamlined and reproducible approach to size-based DNA fractionation, facilitating downstream applications such as genome assembly and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Genelute bacterial genomic dna kit, by Merck Group (2 mentions)
Rs 2, by Pacific Biosciences (2 mentions)
Smrtbell template prep kit 1, by Pacific Biosciences (2 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
G tube, by Covaris (1 mentions)
Smrt cell v3, by Pacific Biosciences (1 mentions)
Smrt analysis 2, by Pacific Biosciences (1 mentions)
Agilent 2200 tapestation, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna br assay, by Thermo Fisher Scientific (1 mentions)
Smarter polymerase chain reaction cdna synthesis kit, by Takara Bio (1 mentions)
Nextera xt dna preparation kit, by Illumina (1 mentions)
Nextseq platform, by Illumina (1 mentions)
5 protocols using bluepippin size selection system protocol
1
Whole-Genome Sequencing of Bacterial Isolate
2
High-Molecular-Weight Parasite DNA Sequencing
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Sequencing libraries were produced using the PacBio 20-kb library preparation protocol for high-molecular-weight gDNA obtained from clonal parasite lines, as previously described (28 (link)). We used the SMRTbell template prep kit 1.0 (Pacific Biosciences) following the standard 20-kb template preparation using the BluePippin size Selection system protocol (Pacific Biosciences). Briefly, parasite DNA was sheared twice for 1 min at 5,300 rpm in an Eppendorf 5424 centrifuge using a g-Tube (Covaris) followed by damage repair, end repair, and ligation of SMARTbell adapters. Unligated DNA was digested with exonucleases, and the libraries were size selected using a BluePippin pulsed-field gel electrophoresis instrument (Sage Science) to isolate fragments greater than 15 kb. Library concentration was measured with the Qubit fluorometer dsDNA BR assay kit (Life Technologies, Inc.), and fragment length distributions were generated using the 2200 TapeStation (Agilent). Sequencing primer and P6 polymerase were annealed to the libraries according to the manufacturer’s protocols (Pacific Biosciences) and performed with P6-C5 chemistry and v3 SMRT cells on an RSII instrument at Weill Cornell Medicine.
3
Single Molecule Sequencing and Genome Assembly
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Single Molecule, Real-Time (SMRT) sequencing libraries were prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) and 20 kb Template Preparation Using BluePippin Size-Selection System protocol (Pacific Biosciences). Library quality and quantity were determined using an Agilent 2200 TapeStation and Qubit dsDNA BR Assay (Life Technologies), respectively. Sequencing was conducted using P5-C3 chemistry and a v3 SMRT Cell (Pacific Biosciences) at Weill Cornell Medical College. Genome assembly was conducted using the Hierarchical Genome Assembly Process 2.0 (HGAP 2.0) (Chin et al. 2013 (link)). Raw sequencing reads were filtered for length and quality such that the minimum polymerase read score was 0.8, the minimum subread length was 500 bp, and the minimum polymerase read score was greater than 100. The assembly was generated using CeleraAssembler v1 with the default parameters and was polished using the Quiver algorithm (Chin et al. 2013 (link)). To identify methylated DNA bases, we used the RS Modification and Motif Analysis module of Pacific Biosciences’ SMRT Analysis 2.3 with a Quality Value (QV) threshold of 30. This analysis uses an in silico kinetic reference and a t-test-based kinetic score to detect modified bases (Flusberg et al. 2010 (link)).
4
Full-length Transcriptome Profiling via PacBio Sequencing
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In order to obtain the complete information of full-length transcriptome, total RNA from 19 different samples were pooled together in an equal quantity to construct libraries for PacBio sequencing. The mixed RNA sample was reverse transcribed using the Clontech SMARTer polymerase chain reaction (PCR) cDNA Synthesis Kit and oligo (dT). Large-scale PCR amplification was carried out to generate barcode full-length cDNA. The BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100-092-800-03) was used to separate the size of PCR selection for mix sample >4 kb. After damage repairing and end joining, the full-length cDNA is ligated to the SMRT dump bell joint. These cDNA products were purified for Iso-Seq SMRTBell library preparation. A SMRT cells were sequenced on the PacBio Sequel System platform.
5
Whole-Genome Sequencing of Bacterial Isolates
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All study isolates underwent whole-genome sequencing (WGS). DNA libraries were prepared using the Nextera XT DNA preparation kit (Illumina), and 2 × 150-bp paired-end sequencing was performed using the NextSeq platform (Illumina), as previously described (64 (link)). One isolate (AR01/DG) underwent genomic DNA extraction with the GenElute bacterial genomic DNA kit (Sigma-Aldrich), followed by sequencing on the RSII (Pacific Biosciences) with P6-C4 chemistry according to the 20-kb template preparation using the BluePippin size selection system protocol (Pacific Biosciences).
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