Predictors of sequence capture in a large-scale anchored phylogenomics project
Predictors of sequence capture in a large-scale anchored phylogenomics project
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DOI：
10.3389/fevo.2022.943361
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发表时间：
2022-11
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影响因子：
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通讯作者：
Renato Nunes;Caroline G. Storer;Tenzing Doleck;A. Kawahara;N. Pierce;David J. Lohman
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作者：
Renato Nunes;Caroline G. Storer;Tenzing Doleck;A. Kawahara;N. Pierce;David J. Lohman
下一代测序（NGS）技术通过降低从多个标记或全基因组生成序列数据所需的成本和时间，彻底改变了系统基因组学。此外，几十年前收集的生物标本的片段DNA可以用NGS测序，减少了收集新标本的需要。序列捕获，也称为锚定杂交富集，是一种为NGS测序产生减少表示库的方法。该技术使用单链寡核苷酸探针与通过NGS测序的预先选择的基因组区域杂交，最终形成来自多个分类群的许多同源位点的数据集。使用这些序列进行系统发育分析有可能解决深层和浅层系统发育关系。确定影响序列捕获成功的因素可以节省时间、金钱和宝贵的标本，尽管测序成功的可能性很低，但这些标本可能被破坏性地取样。在一个大型蝴蝶系统基因组学项目中，我们研究了标本年龄、保存方法和DNA浓度对序列捕获（捕获序列数量和序列质量）的影响，同时考虑了分类和提取的组织类型。本项目使用两组探针从6000多个蝴蝶标本中提取了391个位点或13个位点子集。我们发现序列捕获是一种弹性方法，能够在不同年龄（0-111年）、保存方法（酒精、纸、钉）和DNA浓度（0.020 ng/μl - 316 ng/ul）的样品中扩增位点。回归分析表明，序列捕获与DNA浓度呈正相关。然而，序列捕获和DNA浓度与样品年龄和保存方法呈负相关。我们的研究结果表明，序列捕获项目应优先使用年龄小于20岁的酒精保存样本。在没有这种标本的情况下，任何年龄的干燥样本都可以获得序列数据，尽管其回报随着年龄的增长而减少。
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.