A 10-kb library was prepared and processed as recommended by Pacific Biosciences (http://www.smrtcommunity.com/SampleNet/Sample-Prep ). Library construction and subsequent sequencing were performed using the SMRTbell Template Preparation Reagent Kit 1.0, DNA/Polymerase binding kit P4-C2, MagBead Kit and DNA Sequencing Kit 2.0 (all components supplied by Pacific Biosciences, Menlo Park CA, USA.). The genome was sequenced using PacBio RS II technology (P4-C2 chemistry). Data collected on the PacBio RS II instrument were processed and filtered (SMRT analysis software, version 2.1). All experiments were conducted according to the manufacturers’ instructions on a single SMRT cell. Obtained data were analysed on the SMRT Portal V2.1.1 (http://www.pacb.com/devnet/ ) by applying the integrated Celera® Assembler. SMRT sequencing and SBS were performed by the Max Planck-Genome-centre Cologne, Germany (http://mpgc.mpipz.mpg.de/home/ ).
Dna sequencing kit 2
Manufactured by Pacific Biosciences
Sourced in United States
The DNA Sequencing Kit 2.0 is a laboratory equipment product designed for DNA sequencing. It provides the necessary reagents and consumables required to perform DNA sequencing analysis. The core function of this kit is to enable the determination of the precise order of nucleotides within a DNA molecule.
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Lab products found in correlation
3 protocols using dna sequencing kit 2
1
PacBio Sequencing of 10-kb Library
2
High-Quality Genome Assembly with PacBio
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Twenty micrograms of gDNA was processed to create SMRTbell sequencing templates > 10 kb (average insert size 17 kb) and sequenced using a PacBio RS II System in which polymerase-MagBead-bound templates were loaded at an on-plate concentration of 150 pM. Templates were subsequently sequenced using DNA Sequencing Kit 2.0, with data collection of 180 mins (Pacific Biosciences). Genomes were assembled using HGAP (Chin et al. 2013 (link)) with default parameters in SMRT Analysis Suite version 2.1 (Pacific Biosciences). Additional manual assembly of contigs was carried out in cases of unique overlapping sequence. Consensus sequence polishing was done using the Quiver algorithm in Genomic Consensus version 0.7.0. Base modification analysis was performed by mapping SMRT sequencing reads to the respective assemblies using the BLASR mapper (Chaisson and Tesler 2012 (link)) and SMRT Analysis Suite version 2.1 using standard mapping protocols. Clustering of sequence motifs was performed using Motif Finder (https://github.com/PacificBiosciences/DevNet/wiki/Motiffinder ). See Supplemental Methods for further details.
3
PacBio Sequel Single-Molecule Sequencing
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DNA library was constructed using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, USA), and each constructed library was combined with a primer and DNA polymerase to form a primer/template/polymerase, using the DNA Polymerase Binding Kit P4 (Pacific Biosciences). All complexes were loaded onto the SMRT Cell using the MagBead kit (Pacific Biosciences), and the MagBead was combined with the complex to travel over the zero-mode waveguide (ZMW). Sequencing was performed using the DNA Sequencing Kit 2.0 (Pacific Biosciences) on a PacBio Sequel Sequencer (Pacific Biosciences). The biotin-modified DNA polymerase bound to streptavidin at the bottom of the ZMW, anchoring the complex. The DNA polymerase read the single-stranded circular DNA template several times to produce polymerase sequences.
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