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...
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Published in | PloS one Vol. 10; no. 4; p. e0124671 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
22.04.2015
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: DCM MB. Performed the experiments: DCM MLC. Analyzed the data: DCM. Contributed reagents/materials/analysis tools: MB. Wrote the paper: DCM MB. Edited the manuscript: DCM MB MLC. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0124671 |