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Next-generation sequencing (NGS) technologies have revolutionized phylogenomics by decreasing the cost and time required to generate sequence data from multiple markers or whole genomes. Further, the fragmented DNA of biological specimens collected decades ago can be sequenced with NGS, reducing the need for collecting fresh specimens. Sequence capture, also known as anchored hybrid enrichment, is a method to produce reduced representation libraries for NGS sequencing. The technique uses single-stranded oligonucleotide probes that hybridize with pre-selected regions of the genome that are sequenced via NGS, culminating in a dataset of numerous orthologous loci from multiple taxa. Phylogenetic analyses using these sequences have the potential to resolve deep and shallow phylogenetic relationships. Identifying the factors that affect sequence capture success could save time, money, and valuable specimens that might be destructively sampled despite low likelihood of sequencing success. We investigated the impacts of specimen age, preservation method, and DNA concentration on sequence capture (number of captured sequences and sequence quality) while accounting for taxonomy and extracted tissue type in a large-scale butterfly phylogenomics project. This project used two probe sets to extract 391 loci or a subset of 13 loci from over 6,000 butterfly specimens. We found that sequence capture is a resilient method capable of amplifying loci in samples of varying age (0–111 years), preservation method (alcohol, papered, pinned), and DNA concentration (0.020 ng/μl - 316 ng/ul). Regression analyses demonstrate that sequence capture is positively correlated with DNA concentration. However, sequence capture and DNA concentration are negatively correlated with sample age and preservation method. Our findings suggest that sequence capture projects should prioritize the use of alcohol-preserved samples younger than 20 years old when available. In the absence of such specimens, dried samples of any age can yield sequence data, albeit with returns that diminish with increasing age.
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The Effects of Herbarium Specimen Characteristics on Short-Read NGS Sequencing Success in Nearly 8000 Specimens: Old, Degraded Samples Have Lower DNA Yields but Consistent Sequencing Success
Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies.
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- Award ID(s):
- 1916632
- PAR ID:
- 10311924
- Date Published:
- Journal Name:
- Frontiers in Plant Science
- Volume:
- 12
- ISSN:
- 1664-462X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fpls.2021.669064
