From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows
MetadataShow full item record
Funding and Sponsorship
This open access article is distributed under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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.
Showing items related by title, author, creator and subject.
Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in AcaciaWilliams, A.; Miller, J.; Small, I.; Nevill, Paul; Boykin, L. (2016)Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation ...
Sequencing the ant fauna of a small island: Can metagenomic analysis enable faster identification for routine ant surveys?Majer, Jonathan; Castalanelli, M.; Ledger, J.; Gunawardene, Nihara; Heterick, Brian (2018)All known ant species from a small Western Australian island were subjected to DNA barcoding of the CO1 gene, with a view to using the database to identify ants by Next Generation Sequencing in subsequent, routine surveys. ...
Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety ConcernsCoghlan, Megan; Haile, James; Houston, J.; Murray, D.; White, Nicole; Moolhuijzen, P.; Bellgard, M.; Bunce, Michael (2012)Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, ...