Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing
Abstract Background Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequ...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-01-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-018-5381-7 |
| _version_ | 1818025146109132800 |
|---|---|
| author | Sarah Goldstein Lidia Beka Joerg Graf Jonathan L. Klassen |
| author_facet | Sarah Goldstein Lidia Beka Joerg Graf Jonathan L. Klassen |
| author_sort | Sarah Goldstein |
| collection | DOAJ |
| description | Abstract Background Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. Results We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. Conclusions Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery. |
| first_indexed | 2024-12-10T04:11:28Z |
| format | Article |
| id | doaj.art-84ef08d03b7d4a39ad982cb8ac57992b |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-10T04:11:28Z |
| publishDate | 2019-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-84ef08d03b7d4a39ad982cb8ac57992b2022-12-22T02:02:43ZengBMCBMC Genomics1471-21642019-01-0120111710.1186/s12864-018-5381-7Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencingSarah Goldstein0Lidia Beka1Joerg Graf2Jonathan L. Klassen3Department of Molecular and Cell Biology, University of ConnecticutDepartment of Molecular and Cell Biology, University of ConnecticutDepartment of Molecular and Cell Biology, University of ConnecticutDepartment of Molecular and Cell Biology, University of ConnecticutAbstract Background Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. Results We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. Conclusions Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.http://link.springer.com/article/10.1186/s12864-018-5381-7Oxford Nanopore MinIONGenome sequencingGenome assemblySecondary metabolitesInsertion sequences |
| spellingShingle | Sarah Goldstein Lidia Beka Joerg Graf Jonathan L. Klassen Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing BMC Genomics Oxford Nanopore MinION Genome sequencing Genome assembly Secondary metabolites Insertion sequences |
| title | Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing |
| title_full | Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing |
| title_fullStr | Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing |
| title_full_unstemmed | Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing |
| title_short | Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing |
| title_sort | evaluation of strategies for the assembly of diverse bacterial genomes using minion long read sequencing |
| topic | Oxford Nanopore MinION Genome sequencing Genome assembly Secondary metabolites Insertion sequences |
| url | http://link.springer.com/article/10.1186/s12864-018-5381-7 |
| work_keys_str_mv | AT sarahgoldstein evaluationofstrategiesfortheassemblyofdiversebacterialgenomesusingminionlongreadsequencing AT lidiabeka evaluationofstrategiesfortheassemblyofdiversebacterialgenomesusingminionlongreadsequencing AT joerggraf evaluationofstrategiesfortheassemblyofdiversebacterialgenomesusingminionlongreadsequencing AT jonathanlklassen evaluationofstrategiesfortheassemblyofdiversebacterialgenomesusingminionlongreadsequencing |