Intraspecific DNA contamination distorts subtle population structure in a marine fish: decontamination of herring samples before restriction-site associated (RAD) sequencing and its effects on population genetic statistics

Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies, and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (i) the efficacy of bleach decontamination, (ii) the effect of decontamination on RAD genotypes, and (iii) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and non-spawning (uncontaminated) herring. Samples were soaked for 10 minutes in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction-site associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes, and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high-quality sequences, reproducible genotypes, and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high-throughput sequencing data, and can impact downstream analyses.

野生标本多采集于严苛的野外环境，此时样本易被同物种个体的DNA污染。此类污染不仅会引发难以检出的假基因型分型错误，还可能导致杂合度、等位基因频率及遗传分化的估算失准。体外排放产卵的海洋生物尤易受此影响，究其原因，这类生物的种群遗传分化程度较低，且样本常以批量方式采集，有时甚至取自活跃的产卵集群。本研究以受污染与未受污染的太平洋鲱（Clupea pallasi）样本为材料，开展三项测试：其一为漂白消毒法的去污效能；其二为消毒处理对限制性酶切位点关联DNA（Restriction-site associated DNA, RAD）基因型分型的影响；其三为受污染样本对种群遗传分析的作用后果。我们分别从活跃产卵（因而受污染）的野生太平洋鲱，以及非产卵（未受污染）的个体中采集鳍组织样本，将样本置于漂白液中浸泡10分钟或不作任何处理；随后利用提取得到的DNA，通过限制性酶切位点关联DNA（RAD）建库法构建DNA文库。研究结果显示，种内DNA污染会干扰个体与种群的变异模式，导致杂合子过剩，并对种群结构的估算造成偏差。漂白消毒法可有效去除种内DNA污染，且与RAD测序技术兼容，能够获得高质量序列、可重复的基因型分型结果，同时缺失数据占比极低。尽管精子污染可能仅见于体外排放产卵类生物，但样本的种内污染现象或普遍存在，且难以从高通量测序数据中检出，进而对后续分析产生影响。