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A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation
Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article des...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811786/ https://www.ncbi.nlm.nih.gov/pubmed/33520432 http://dx.doi.org/10.7717/peerj.10420 |
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author | D’Ercole, Jacopo Prosser, Sean W.J. Hebert, Paul D.N. |
author_facet | D’Ercole, Jacopo Prosser, Sean W.J. Hebert, Paul D.N. |
author_sort | D’Ercole, Jacopo |
collection | PubMed |
description | Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E−4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible. |
format | Online Article Text |
id | pubmed-7811786 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-78117862021-01-28 A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation D’Ercole, Jacopo Prosser, Sean W.J. Hebert, Paul D.N. PeerJ Biodiversity Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E−4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible. PeerJ Inc. 2021-01-14 /pmc/articles/PMC7811786/ /pubmed/33520432 http://dx.doi.org/10.7717/peerj.10420 Text en © 2021 D’Ercole et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Biodiversity D’Ercole, Jacopo Prosser, Sean W.J. Hebert, Paul D.N. A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title | A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title_full | A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title_fullStr | A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title_full_unstemmed | A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title_short | A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation |
title_sort | smrt approach for targeted amplicon sequencing of museum specimens (lepidoptera)—patterns of nucleotide misincorporation |
topic | Biodiversity |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811786/ https://www.ncbi.nlm.nih.gov/pubmed/33520432 http://dx.doi.org/10.7717/peerj.10420 |
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