Intraspecific genetic variation in complex assemblages from mitochondrial metagenomics: comparison with DNA barcodes
Intraspecific genetic variation in complex assemblages from mitochondrial metagenomics: comparison with DNA barcodes
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DOI：
10.1111/2041-210x.12667
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发表时间：
2017-02-01
影响因子：
6.6
通讯作者：
Vogler, Alfried P.
中科院分区：
文献类型：
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作者：
Gomez-Rodriguez, Carola;Timmermans, Martijn J. T. N.;Vogler, Alfried P.
1.使用Illumina技术进行后基因组鸟枪测序，并对现场采集的混合无脊椎动物样本进行从头组装，很容易产生线粒体基因组序列，从而能够快速识别和量化物种多样性。然而，样本池中存在的种内遗传变异性在有丝分裂组组装过程中丢失，这限制了线粒体元基因组学在种群多样性研究中的应用。利用叶甲科(叶甲科)的10个自然群落(>；2600个个体)，比较了Sanger测序个体和Illumina鸟枪测序样本库线粒体cox1-5条形码的DNA变异。一般来说，每个物种只组装一个线粒体重叠群，即使存在种内变异。忽略使用两个不同的组装者的歧义性，来自这些组装体的cox1条形码区域在907%的案例中与Sanger测序的条形码完全匹配，而在具有较大种内和种间变异性的样本的组装体中，这一比例下降到76%。两种数据类型的条形码之间的核苷酸差异几乎完全在同义的第三密码子位置，尽管受影响的位点数量非常少，并且最大的差异与Sanger序列的质量较差相关。未组装的鸟枪读数也被用来对单核苷酸多态进行评分，并计算每个地点所有可用种群的种内核苷酸多样性(Pi)。这些值与Sanger测序的cox1变异相关，但显著高于Sanger测序的cox1变异。总体而言，集中于集合样本的鸟枪测序产生的核苷酸变异数据与公认的专注于样本的Sanger方法相当。因此，这些发现扩大了复杂生物多样性样本的线粒体元基因组学在物种水平以下的多样性评估中的应用。
1. Metagenomic shotgun sequencing, using Illumina technology, and de novo genome assembly of mixed field-collected samples of invertebrates readily produce mitochondrial genome sequences, allowing rapid identification and quantification of species diversity. However, intraspecific genetic variability present in the specimen pools is lost during mitogenome assembly, which limits the utility of mitochondrial metagenomics' for studies of population diversity. Using 10 natural communities (>2600 individuals) of leaf beetles (Chrysomelidae), DNA variation in the mitochondrial cox1-5 barcode' was compared for Sanger-sequenced individuals and Illumina shotgun-sequenced specimen pools. Generally, only a single mitochondrial contig was assembled per species, even in the presence of intraspecific variation. Ignoring ambiguity from the use of two different assemblers, the cox1 barcode regions from these assemblies were exact nucleotide matches of a Sanger-sequenced barcode in 907% of cases, which dropped to 76% in assemblies from samples with large intra- and interspecific variability. Nucleotide differences between barcodes from both data types were almost exclusively in synonymous 3rd codon positions, although the number of affected sites was very low, and the greatest discrepancies were correlated with poor quality of Sanger sequences. Unassembled shotgun reads were also used to score single nucleotide polymorphisms and to calculate intraspecific nucleotide diversity (pi) for all available populations at each site. These values correlated with Sanger-sequenced cox1 variation but were significantly higher. Overall, the assemblage-focused shotgun sequencing of pooled samples produced nucleotide variation data comparable to the well-established specimen-focused Sanger approach. The findings thus extend the application of mitochondrial metagenomics of complex biodiversity samples to the estimation of diversity below the species level.