Data from: Assessing the utility of whole genome amplified DNA for next-generation molecular ecology

DNA quantity can be a hindrance in ecological and evolutionary research programs due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next-generation sequencing of non-model taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double-digest RAD sequencing on the gray mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de-novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de-novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next-generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.

在生态学与进化研究中，DNA量不足常因多种因素成为阻碍，包括目标物种的濒危状态、可获取的组织类型，以及野外条件对样本保存方式的影响。针对低量DNA样本，全基因组扩增（whole genome amplification, WGA）技术可显著提升DNA产出量，是潜在的解决方案。迄今为止，鲜有研究严格评估非模式类群经全基因组扩增与下一代测序后可能产生的序列偏倚。为填补这一认知空白，本研究以灰鼠狐猴（Microcebus murinus）为研究对象，采用多重置换扩增（multiple displacement amplification, MDA）与双酶切RAD测序技术，对比原始基因组DNA（genomic DNA, gDNA），量化基因组覆盖度与单核苷酸多态性（single nucleotide polymorphism, SNP）位点调用过程中的偏倚情况。本研究严格遵循试剂盒厂商推荐的起始DNA量（>100 ng）标准，旨在为潜在偏倚提供基线评估数据。研究结果强烈表明，多重置换扩增富集不会对基因组特征分析引入系统性偏倚。当设定基因型调用的最低阈值为20X测序深度时，无论是基于从头组装还是参考基因组进行基因分型，样本间的SNP位点调用一致性均高达98%以上。MDA与gDNA的相对基因组覆盖度亦无显著差异，且未检测到等位基因缺失现象。SNP位点一致性随测序深度阈值变化而改变：基于从头组装进行基因分型时，约12X测序深度即可达到95%的一致性；而基于参考基因组进行基因分型时，该阈值约为7X。上述结果表明，当DNA量成为分子生态学研究的限制因素时，多重置换扩增可作为下一代分子生态学研究的合适解决方案。