Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device
Abstract Background Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expens...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-03-01
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| Series: | BMC Bioinformatics |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12859-023-05226-y |
| _version_ | 1797859852009078784 |
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| author | Scott D. Brown Lisa Dreolini Jessica F. Wilson Miruna Balasundaram Robert A. Holt |
| author_facet | Scott D. Brown Lisa Dreolini Jessica F. Wilson Miruna Balasundaram Robert A. Holt |
| author_sort | Scott D. Brown |
| collection | DOAJ |
| description | Abstract Background Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. Results Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. Conclusions Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required. |
| first_indexed | 2024-04-09T21:36:18Z |
| format | Article |
| id | doaj.art-dc10f57d9bd44154b770c27724b9f67e |
| institution | Directory Open Access Journal |
| issn | 1471-2105 |
| language | English |
| last_indexed | 2024-04-09T21:36:18Z |
| publishDate | 2023-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Bioinformatics |
| spelling | doaj.art-dc10f57d9bd44154b770c27724b9f67e2023-03-26T11:18:44ZengBMCBMC Bioinformatics1471-21052023-03-0124111610.1186/s12859-023-05226-yComplete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION deviceScott D. Brown0Lisa Dreolini1Jessica F. Wilson2Miruna Balasundaram3Robert A. Holt4Canada’s Michael Smith Genome Sciences Centre, BC Cancer Research InstituteCanada’s Michael Smith Genome Sciences Centre, BC Cancer Research InstituteCanada’s Michael Smith Genome Sciences Centre, BC Cancer Research InstituteCanada’s Michael Smith Genome Sciences Centre, BC Cancer Research InstituteCanada’s Michael Smith Genome Sciences Centre, BC Cancer Research InstituteAbstract Background Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. Results Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. Conclusions Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.https://doi.org/10.1186/s12859-023-05226-yMinIONCell therapyPlasmid sequencingLong-read sequencing |
| spellingShingle | Scott D. Brown Lisa Dreolini Jessica F. Wilson Miruna Balasundaram Robert A. Holt Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device BMC Bioinformatics MinION Cell therapy Plasmid sequencing Long-read sequencing |
| title | Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device |
| title_full | Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device |
| title_fullStr | Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device |
| title_full_unstemmed | Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device |
| title_short | Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device |
| title_sort | complete sequence verification of plasmid dna using the oxford nanopore technologies minion device |
| topic | MinION Cell therapy Plasmid sequencing Long-read sequencing |
| url | https://doi.org/10.1186/s12859-023-05226-y |
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