A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2017 Aug 4

PMID 28769883

Citations 17

Authors

Sagar M Utturkar

Dawn M Klingeman

Richard A Hurt Jr

Steven D Brown

Affiliations

Soon will be listed here.

Abstract

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

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References

Coil D, Jospin G, Darling A . A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data. Bioinformatics. 2014; 31(4):587-9. DOI: 10.1093/bioinformatics/btu661. View

Brown S, Nagaraju S, Utturkar S, Tissera S, Segovia S, Mitchell W . Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia. Biotechnol Biofuels. 2014; 7:40. PMC: 4022347. DOI: 10.1186/1754-6834-7-40. View

Utturkar S, Klingeman D, Bruno-Barcena J, Chinn M, Grunden A, Kopke M . Sequence data for Clostridium autoethanogenum using three generations of sequencing technologies. Sci Data. 2015; 2:150014. PMC: 4409012. DOI: 10.1038/sdata.2015.14. View

Satou K, Shiroma A, Teruya K, Shimoji M, Nakano K, Juan A . Complete Genome Sequences of Eight Helicobacter pylori Strains with Different Virulence Factor Genotypes and Methylation Profiles, Isolated from Patients with Diverse Gastrointestinal Diseases on Okinawa Island, Japan, Determined Using PacBio.... Genome Announc. 2014; 2(2). PMC: 3990747. DOI: 10.1128/genomeA.00286-14. View

Kanda K, Nakashima K, Nagano Y . Complete Genome Sequence of Bacillus thuringiensis Serovar Tolworthi Strain Pasteur Institute Standard. Genome Announc. 2015; 3(4). PMC: 4490846. DOI: 10.1128/genomeA.00710-15. View

Risse J, Thomson M, Patrick S, Blakely G, Koutsovoulos G, Blaxter M . A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data. Gigascience. 2015; 4:60. PMC: 4670535. DOI: 10.1186/s13742-015-0101-6. View

Otto T, Sanders M, Berriman M, Newbold C . Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology. Bioinformatics. 2010; 26(14):1704-7. PMC: 2894513. DOI: 10.1093/bioinformatics/btq269. View

Lancaster W, Utturkar S, Poole F, Klingeman D, Elias D, Adams M . Near-Complete Genome Sequence of Clostridium paradoxum Strain JW-YL-7. Genome Announc. 2016; 4(3). PMC: 4859166. DOI: 10.1128/genomeA.00229-16. View

Woo H, Utturkar S, Klingeman D, Simmons B, DeAngelis K, Brown S . Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil. Genome Announc. 2014; 2(3). PMC: 4064042. DOI: 10.1128/genomeA.00637-14. View

10.

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S . Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12):1647-9. PMC: 3371832. DOI: 10.1093/bioinformatics/bts199. View

11.

Roberts R, Carneiro M, Schatz M . The advantages of SMRT sequencing. Genome Biol. 2013; 14(7):405. PMC: 3953343. DOI: 10.1186/gb-2013-14-6-405. View

12.

Koren S, Phillippy A . One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly. Curr Opin Microbiol. 2014; 23:110-20. DOI: 10.1016/j.mib.2014.11.014. View

13.

Deschamps S, Mudge J, Cameron C, Ramaraj T, Anand A, Fengler K . Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens. Sci Rep. 2016; 6:28625. PMC: 4923883. DOI: 10.1038/srep28625. View

14.

Feng Y, Zhang Y, Ying C, Wang D, Du C . Nanopore-based fourth-generation DNA sequencing technology. Genomics Proteomics Bioinformatics. 2015; 13(1):4-16. PMC: 4411503. DOI: 10.1016/j.gpb.2015.01.009. View

15.

Chin C, Alexander D, Marks P, Klammer A, Drake J, Heiner C . Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. 2013; 10(6):563-9. DOI: 10.1038/nmeth.2474. View

16.

Quail M, Smith M, Coupland P, Otto T, Harris S, Connor T . A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012; 13:341. PMC: 3431227. DOI: 10.1186/1471-2164-13-341. View

17.

Brown S, Utturkar S, Magnuson T, Ray A, Poole F, Lancaster W . Complete Genome Sequence of Pelosinus sp. Strain UFO1 Assembled Using Single-Molecule Real-Time DNA Sequencing Technology. Genome Announc. 2014; 2(5). PMC: 4155594. DOI: 10.1128/genomeA.00881-14. View

18.

Treangen T, Salzberg S . Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet. 2011; 13(1):36-46. PMC: 3324860. DOI: 10.1038/nrg3117. View

19.

Shapiro L, Scully E, Roberts D, Straub T, Geib S, Park J . Draft Genome Sequence of Erwinia tracheiphila, an Economically Important Bacterial Pathogen of Cucurbits. Genome Announc. 2015; 3(3). PMC: 4457052. DOI: 10.1128/genomeA.00482-15. View

20.

Page A, De Silva N, Hunt M, Quail M, Parkhill J, Harris S . Robust high-throughput prokaryote assembly and improvement pipeline for Illumina data. Microb Genom. 2017; 2(8):e000083. PMC: 5320598. DOI: 10.1099/mgen.0.000083. View