A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies
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. Furthermore, 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.
- Authors:
-
- Univ. of Tennessee, Knoxville, TN (United States); Purdue Univ., West Lafayette, IN (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); BioEnergy Science Center, Oak Ridge, TN (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); BioEnergy Science Center, Oak Ridge, TN (United States)
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1376350
- Grant/Contract Number:
- AC05-00OR22725
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Frontiers in Microbiology
- Additional Journal Information:
- Journal Volume: 8; Journal ID: ISSN 1664-302X
- Publisher:
- Frontiers Research Foundation
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; PacBio; Illumina; genome assembly; next-generation sequencing (NGS); repetitive DNA; Pilon; circlator
Citation Formats
Utturkar, Sagar M., Klingeman, Dawn M., Hurt, Jr., Richard A., and Brown, Steven D. A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies. United States: N. p., 2017.
Web. doi:10.3389/fmicb.2017.01272.
Utturkar, Sagar M., Klingeman, Dawn M., Hurt, Jr., Richard A., & Brown, Steven D. A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies. United States. https://doi.org/10.3389/fmicb.2017.01272
Utturkar, Sagar M., Klingeman, Dawn M., Hurt, Jr., Richard A., and Brown, Steven D. Tue .
"A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies". United States. https://doi.org/10.3389/fmicb.2017.01272. https://www.osti.gov/servlets/purl/1376350.
@article{osti_1376350,
title = {A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies},
author = {Utturkar, Sagar M. and Klingeman, Dawn M. and Hurt, Jr., Richard A. and Brown, Steven D.},
abstractNote = {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. Furthermore, 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.},
doi = {10.3389/fmicb.2017.01272},
journal = {Frontiers in Microbiology},
number = ,
volume = 8,
place = {United States},
year = {Tue Jul 18 00:00:00 EDT 2017},
month = {Tue Jul 18 00:00:00 EDT 2017}
}
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- Schein, Catherine H.
- Molecular Neurobiology, Vol. 56, Issue 5
Comparative genomic analysis of monosporidial and monoteliosporic cultures for unraveling the complexity of molecular pathogenesis of Tilletia indica pathogen of wheat
journal, June 2019
- Mishra, Pallavi; Maurya, Ranjeet; Gupta, Vijai K.
- Scientific Reports, Vol. 9, Issue 1
Graph analysis of fragmented long-read bacterial genome assemblies
journal, March 2019
- Marijon, Pierre; Chikhi, Rayan; Varré, Jean-Stéphane
- Bioinformatics, Vol. 35, Issue 21
Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats
journal, August 2018
- Schmid, Michael; Frei, Daniel; Patrignani, Andrea
- Nucleic Acids Research, Vol. 46, Issue 17
Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains
journal, January 2018
- Acuña-Amador, Luis; Primot, Aline; Cadieu, Edouard
- BMC Genomics, Vol. 19, Issue 1
Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4
journal, January 2019
- Gan, Han Ming; Lee, Melvin V. L.; Savka, Michael A.
- PeerJ, Vol. 7
Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats
text, January 2018
- Schmid, Michael; Frei, Daniel; Patrignani, Andrea
- Oxford University Press
Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats
text, January 2018
- Schmid, Michael; Frei, Daniel; Patrignani, Andrea
- ETH Zurich
Polyglutamine Repeats in Viruses
journal, September 2018
- Schein, Catherine H.
- Molecular Neurobiology, Vol. 56, Issue 5
Comparative genomic analysis of monosporidial and monoteliosporic cultures for unraveling the complexity of molecular pathogenesis of Tilletia indica pathogen of wheat
journal, June 2019
- Mishra, Pallavi; Maurya, Ranjeet; Gupta, Vijai K.
- Scientific Reports, Vol. 9, Issue 1
Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats
text, January 2018
- Schmid, Michael; Frei, Daniel; Patrignani, Andrea
- ETH Zurich
Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4
journal, January 2019
- Gan, Han Ming; Lee, Melvin V. L.; Savka, Michael A.
- PeerJ, Vol. 7