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A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies
Sagar M Utturkar1, Dawn M Klingeman2,3, Richard A Hurt2
1Graduate School of Genome Science and Technology, University of TennesseeKnoxville, TN, United States.
Frontiers in Microbiology
|August 4, 2017
Summary
This study analyzed unassembled DNA in bacterial genomes sequenced by PacBio and Illumina technologies. Repetitive DNA and low coverage, not secondary structures, primarily caused assembly gaps, aiding future microbial genome finishing.
Area of Science:
- Genomics
- Bioinformatics
- Microbiology
Background:
- High-throughput sequencing technologies like PacBio and Illumina are crucial for bacterial genome assembly.
- Unassembled DNA regions (gaps) can hinder complete genome characterization and analysis.
- Understanding the causes of these gaps is essential for improving genome assembly strategies.
Purpose of the Study:
- To characterize DNA regions unassembled by PacBio and Illumina sequencing in seven bacterial genomes.
- To investigate the biological factors contributing to assembly gaps.
- To provide insights for improving microbial genome finishing techniques.
Main Methods:
- Comparative analysis of unassembled regions from PacBio and Illumina sequencing data.
- Manual genome finishing using bioinformatics and PCR/Sanger sequencing for two bacterial genomes.
- Evaluation of PacBio gap sequences for properties like GC content, read coverage, and secondary structure formation.
- Analysis of biological features within gaps, including repetitive DNA, transposons, plasmids, and phage integrations.
Main Results:
- Illumina assembly gaps were primarily associated with repetitive DNA, such as rRNA operons.
- PacBio assembly gaps were attributed to a combination of lower-than-average sequence coverage and repetitive sequences at contig ends.
- Strong DNA secondary structures were not found to be a significant cause of gaps.
- Identified biological factors interfering with assembly included active transposons, multiple plasmids, phage DNA, and large duplications.
Conclusions:
- Genome assembly gaps are influenced by a combination of technical and biological factors.
- PacBio sequencing offers high-quality assemblies with fewer limitations compared to Illumina for gap regions.
- The findings provide a framework for addressing and resolving unassembled DNA in microbial genomics.
- This systematic evaluation aids researchers in closing and polishing microbial genome sequences effectively.
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