Short fragment DNA libraries were generated using the Illumina NexteraXT DNA preparation kit and fragment sequencing was undertaken with the Illumina NextSeq 500 platform using 2 × 150 bp chemistry. Highly intact and high quality genomic DNA was extracted from Ef_aus0233 and subjected to Pacific Biosciences SMRT sequencing according to the manufacturer’s instructions and sequenced with two SMRT cells on the RS II platform (Pacific Biosciences) using P5-C3 chemistry. Genome assembly was performed using the SMRT Analysis System v2.3.0.140936 (Pacific Biosciences). Raw sequence data were de novo assembled using the HGAP v3 protocol with a genome size of 3 Mb. Polished contigs were error corrected using Quiver v1. The resulting assembly was then checked using BridgeMapper v1 in the SMRT Analysis System, and the consensus sequence corrected with short-read Illumina data, using the program Snippy (https://github.com/tseemann/snippy ). The final chromosome assembly was validated by reference to a high-resolution NcoI optical map using MapSolver (version 3.10; OpGen, Gaithersburg, MD, USA). Common bacterial DNA base modifications and methyltransferase motifs were assessed using the protocol, RS_Modification_and_Motif_Analysis in the SMRT Analysis System v2.3.0.140936 (Pacific Biosciences).
Rsii platform
Manufactured by Pacific Biosciences
Sourced in United States
The RSII platform is a DNA sequencing instrument developed by Pacific Biosciences. It utilizes single-molecule real-time (SMRT) sequencing technology to perform high-throughput DNA sequencing. The RSII platform is designed for researchers and scientists to conduct a wide range of genomic applications, including de novo genome assembly, targeted sequencing, and epigenetic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna preparation kit, by Illumina (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (3 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Nutrient agar, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Clontech smarter pcr cdna synthesis kit, by Takara Bio (2 mentions)
Bluepippin, by Sage Science (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Dneasy blood and tissue extraction kit, by Qiagen (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Rapid barcoding kit sqk rbk004, by Oxford Nanopore (2 mentions)
Smrt sequencing, by Pacific Biosciences (1 mentions)
Smrt analysis system v2, by Pacific Biosciences (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Dna template prep kit 2, by Pacific Biosciences (1 mentions)
Novaseq 6000, by Pacific Biosciences (1 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
44 protocols using rsii platform
1
Hybrid genome assembly and modification
2
Transcriptome Sequencing of Centipedegrass
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Using the TRIzol kit (Invitrogen, CA, USA) and its instructions, total RNA was extracted from 12 centipedegrass leaf samples. Each sample was electrophoresed on an agarose gel to determine its RNA quality. The RNA purity was determined using a NanoPhotometer® spectrophotometer (IMPLEN, Munich, Germany). Using SMARTer™, the PCR cDNA Synthesis Kit (Pacific Biosciences, San Diego, CA, USA) synthesized the full-length cDNA of mRNA, amplified the full-length cDNA using PCR, repaired the ends of the full-length cDNA, connected the SMRT dumbbell-shaped connector and finally performed exonuclease digestion to obtain the cDNA library. The Pacific Biosciences DNA Template Prep Kit 2.0 was used in the construction of SMRTbell iso-seq libraries. Sequencing was performed on the RS II platform (Pacific Biosciences, San Diego, CA, USA). To construct the cDNA library for SGS, the manufacturer provided a protocol for using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Beverly, MA, USA). SGS produced 125 bp paired-end sequence reads (2 × 125 bp) based on qualified libraries applied to Illumina’s NovaSeq 6000 (Pacific Biosciences, San Diego, CA, USA). The Biomarker Technology Co. (Beijing, China) performed the high-throughput sequencing (TGS and SGS) for this study.
3
Highly Contiguous A4 Genome Assembly
4
Salting-out DNA Extraction and SMRT Sequencing
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The salting-out method (47 ) was used to extract genomic DNA. The DNA was sequenced at the Earlham Institute (Norwich, UK) using SMRT sequencing technology (Pacific Biosciences RSII platform) and assembled using the HGAP2 pipeline (39 (link)).
5
Whole Genome Sequencing of Bacterial Strains
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The whole genomes of L. britannica R. variigena G. quercinecans B. goodwinii R. victoriana
6
Complete Genome Sequencing of Petroleum-Degrading Bacterium
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The strain was previously isolated from petroleum contaminated soil as described before (Nahurira et al. 2017 (link)). It was cultured in LB liquid medium for 4 days at 30 °C and 180 rpm until the OD600 reached 1.0. This culture was then used for DNA extraction.
The genomic DNA was extracted using Qiagen QIAamp DNA Kit according to manufacturer’s instructions. The size, quantity and quality of the DNA were checked by Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The genome was then sequenced by Pacific Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology (Ardui et al. 2018 (link); Shin et al. 2013 (link)). The raw sequence data were filtered, and 186,554 high quality reads were obtained with a total of 1,912,577,568 bp and average reads length of 10,252 bp. The reads were assembled de novo by applying the hierarchical genome assembly process (HGAP) algorithm version 2 (Jayakumar and Sakakibara 2017 (link)). Prediction and annotation of genes, protein coding sequences, tRNA and rRNA carried out using by NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ ).
The genomic DNA was extracted using Qiagen QIAamp DNA Kit according to manufacturer’s instructions. The size, quantity and quality of the DNA were checked by Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The genome was then sequenced by Pacific Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology (Ardui et al. 2018 (link); Shin et al. 2013 (link)). The raw sequence data were filtered, and 186,554 high quality reads were obtained with a total of 1,912,577,568 bp and average reads length of 10,252 bp. The reads were assembled de novo by applying the hierarchical genome assembly process (HGAP) algorithm version 2 (Jayakumar and Sakakibara 2017 (link)). Prediction and annotation of genes, protein coding sequences, tRNA and rRNA carried out using by NCBI Prokaryotic Genome Annotation Pipeline (
7
Multi-Sequencing Platform Genome Characterization
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DNA was extracted using the phenol chloroform protocol (Neumann et al., 1992 (link)). The genome was sequenced separately in three sequencing runs: (1) Using an Illumina GAIIx platform (72 bp paired-end read). The libraries for this run were produced as follows: DNA was sheared by sonication using a Bio-Ruptor ultrasonicator (Diagenode), end-repaired using the Quick Blunting kit from New England Biolabs, and Illumina adaptors were ligated using the quick ligate kit from Enzymatics followed by PCR enrichment. Size selection was performed using SPRI beads to obtain fragments with a mean size of 228 bp; (2) Using an Illumina HiSeq2000 platform (150 bp paired-end read). The libraries for this sequencing run were produced at the University of Haifa Bioinformatics Support Unit using the NEBNext Ultra DNA Library Prep Kit for Illumina followed by size selection using SPRI beads to obtain fragments with a mean size of 475 bp; (3) Using a Pacific Biosciences RS II platform, with the libraries produced by the DNA template prep kit 3.0 from Pacific Biosciences, at the Yale Center for Genome Analysis.
8
Transcriptome Profiling of Bagrada odoriphaga Life Stages
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SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA) was used to isolate the B. odoriphaga eggs, second instar larvae, fourth instar larvae, pupae, and adult total RNA according to the manufacturer’s protocols, after which possible residual genomic DNA was removed with deoxyribonuclease (DNase I: Fermentas Inc., Burlington, ON, Canada). The RNA integrity was determined using Agilent RNA 6000 Nano Reagents Port 1 (Santa Clara, CA, USA) on a Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) (Table S1 ). Total RNA from different developmental stages was mixed as one sample for cDNA library construction. cDNA synthesis and library construction was conducted using a Clontech SMARTer PCR cDNA Synthesis Kit (catalogue number: 634925) (Clontech, Mountain View, CA, USA). Sequencing was conducted on the Pacific Biosciences RS II platform (Pacific Biosciences, Menlo Park, CA, USA). BluePippin (Sage Science, Beverly, MA, USA) was used for selection of cDNA sequences ranging in size from 1 to 2 kb, 2 to 3 kb, and 3 to 6 kb. Three, three, and two SMRT (Single-molecule real-time sequencing) cells were used to sequence the 1–2 kb, 2–3 kb, and 3–6 kb libraries, respectively, and the reads were deposited in the NCBI (National Center for Biotechnology Information) Sequence Read Archive database under the accession numbers SRR8903502, SRR8903501, and SRR8903503.
9
Complete Genome Sequencing of S. Indiana C629
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The complete genome of S. Indiana C629 was isolated as described by Wang et al. [15 ]. Briefly, whole-genome sequencing was performed using the Pacific Biosciences RS II platform (SMRT® Pacific Biosciences, Menlo Park, CA, USA). De novo assembly of the reads obtained was carried out using continuous long reads (CLR) following the Hierarchical Genome Assembly Process (HGAP) workflow (PacBioDevNet; Pacific Biosciences) as available in the SMRT® Analysis v2.3 program [16 (link)]. The predicted functions of proteins identified were annotated based on homologs when compared to SwissProt (http://www.uniprot.org/uniprot/ ) clusters of orthologs groups (COG) (http://www.ncbi.nlm.nih.gov/COG/ ), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) based on the Best-placed reference protein set and GeneMarkS+. The complete genome of S. Indiana plasmid pC629 was deposited in the NCBI database with the fallowing accession number CP015725 (plasmid pC629).
10
Complete Genome Sequencing of S. schleiferi
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S. schleiferi genomic DNA was purified, sequenced, and assembled into complete genomes as previously described (23 (link)). Briefly, DNA was extracted from overnight cultures of S. schleiferi isolates by using the Qiagen Genomic Tips kit (Valencia, CA, USA). DNA quantity and quality were assessed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Pittsburgh, PA) and a Qubit fluorometer (Life Technologies, Grand Island, NY, USA). Agarose gel electrophoresis was used to confirm high-molecular-weight DNA (>50 kb) for single-molecule real-time sequencing on a Pacific Biosciences RSII platform. SMRTbell adapters were ligated, and each strain of S. schleiferi was sequenced on 1 cell with one 120-min movie. A hierarchical genome assembly process (HGAP) was performed for each strain using the HGAP.3 module (58 (link)). The genome was closed using manual refinement.
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