From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows
Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considera...
| Main Authors: | , , |
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| Format: | Journal Article |
| Published: |
Public Library of Science
2015
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| Online Access: | http://purl.org/au-research/grants/arc/FT0991741 http://hdl.handle.net/20.500.11937/41207 |
| _version_ | 1848756080873570304 |
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| author | Murray, D. Coghlan, Megan Bunce, Michael |
| author_facet | Murray, D. Coghlan, Megan Bunce, Michael |
| author_sort | Murray, D. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose. |
| first_indexed | 2025-11-14T09:06:31Z |
| format | Journal Article |
| id | curtin-20.500.11937-41207 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T09:06:31Z |
| publishDate | 2015 |
| publisher | Public Library of Science |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-412072017-09-13T16:05:05Z From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows Murray, D. Coghlan, Megan Bunce, Michael Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose. 2015 Journal Article http://hdl.handle.net/20.500.11937/41207 10.5061/dryad.2qf0t http://purl.org/au-research/grants/arc/FT0991741 http://purl.org/au-research/grants/arc/DP120103725 Public Library of Science fulltext |
| spellingShingle | Murray, D. Coghlan, Megan Bunce, Michael From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title | From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title_full | From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title_fullStr | From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title_full_unstemmed | From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title_short | From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows |
| title_sort | from benchtop to desktop: important considerations when designing amplicon sequencing workflows |
| url | http://purl.org/au-research/grants/arc/FT0991741 http://purl.org/au-research/grants/arc/FT0991741 http://hdl.handle.net/20.500.11937/41207 |