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Toward Complete Bacterial Genome Sequencing Through the Combined Use of Multiple Next-Generation Sequencing PlatformsS
Toward Complete Bacterial Genome Sequencing Through the Combined Use of Multiple Next-Generation Sequencing PlatformsS
Journal of Microbiology and Biotechnology. 2016. Feb, 26(1): 207-212
Copyright © 2016, The Korean Society For Microbiology And Biotechnology
  • Received : July 15, 2015
  • Accepted : October 14, 2015
  • Published : February 28, 2016
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About the Authors
Haeyoung Jeong
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea
hyjeong@kribb.re.kr
Dae-Hee Lee
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea
Choong-Min Ryu
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea
Seung-Hwan Park
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea

Abstract
PacBio’s long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of second-generation, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.
Keywords
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( A ) LASTZ alignment [10] between two versions of Shigella boydii HGAP assemblies. The upper panel shows a dot plot; and the lower panel, alignment blocks. The major contig from the old version of HGAP is shown in the horizontal axis. The plots were generated using Geneious Pro R8 ( http://www.geneious.com ). ( B ) MUMmer whole-genome alignments [15] of two versions of Paenibacillus sp. HS311 HGAP assemblies (left, old version; right, new version) with the complete genome sequence of P. polymyxa CR1 (upper panel) and cumulative GC skew plots as calculated by (G-C)/(G+C) with a window size of 5 kb (lower panel). ( C ) Ion Torrent PGM mate-pair reads on Pseudomonas syringae pv. syringae HGAP contigs were mapped and visualized using Consed software [8] , the results indicating that the four contigs are arranged in a single scaffold. The lightgreen plot designates the read depth. Multiple copies of ribosomal RNA genes, designated by the thick arrows at the bottom, induced mate reads to align at a longer span (○). RNA genes at the end of the adjacent contigs, represented through filled-in arrows of the same color, were used to join them, resulting in two contigs.
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Acknowledgements
This work was supported by the KRIBB Research Initiative Program, Ministry of Science, ICT, and Future Planning, and by the Next-Generation BioGreen 21 Program (SSAC Grant No. PJ009524) funded by the RDA (to C.M.R), Republic of Korea.
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