High-Quality Genome Assembly with PacBio

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.

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Nandi T., Holden M.T., Didelot X., Mehershahi K., Boddey J.A., Beacham I., Peak I., Harting J., Baybayan P., Guo Y., Wang S., How L.C., Sim B., Essex-Lopresti A., Sarkar-Tyson M., Nelson M., Smither S., Ong C., Aw L.T., Hoon C.H., Michell S., Studholme D.J., Titball R., Chen S.L., Parkhill J, & Tan P. (2015). Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles. Genome Research, 25(1), 129-141.

Publication 2015

RS II System DNA Sequencing Kit 2.0

Chin Consensus sequence Genomic Hgap Mapping protocols Quiver Smrt

Corresponding Organization :

Other organizations : Genome Institute of Singapore, Wellcome Sanger Institute, Imperial College London, National University of Singapore, Griffith University, Pacific Biosciences (United States), Defence Science and Technology Laboratory, DSO National Laboratories, University of Exeter, University of London, London School of Hygiene & Tropical Medicine, Duke-NUS Medical School

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Variable analysis

independent variables

Amount of gDNA processed (20 micrograms)
Method of creating SMRTbell sequencing templates (to create templates > 10 kb with average insert size of 17 kb)
Sequencing platform (PacBio RS II System)
Polymerase-MagBead-bound template loading concentration (150 pM)
Sequencing kit used (DNA Sequencing Kit 2.0)
Data collection duration (180 minutes)
Genome assembly method (HGAP with default parameters in SMRT Analysis Suite version 2.1)
Additional manual assembly of contigs
Consensus sequence polishing method (Quiver algorithm in Genomic Consensus version 0.7.0)
Base modification analysis method (BLASR mapper and SMRT Analysis Suite version 2.1)
Sequence motif clustering method (Motif Finder)

dependent variables

Sequence data generated
Genome assembly quality
Base modifications identified

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the protocol.

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