Digital genotyping of macrosatellites and multicopy genes reveals novel biological functions associated with copy number variation of large tandem repeats.
Digital genotyping of macrosatellites and multicopy genes reveals novel biological functions associated with copy number variation of large tandem repeats.
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DOI：
10.1371/journal.pgen.1004418
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发表时间：
2014-06
期刊：
影响因子：
4.5
通讯作者：
Sharp AJ
中科院分区：
文献类型：
--
作者：
Brahmachary M;Guilmatre A;Quilez J;Hasson D;Borel C;Warburton P;Sharp AJ
串联重复序列在真核生物基因组中很常见，但由于分析困难，对它们的研究仍然很少。在这里，我们展示了Nanostring技术作为一种有针对性的方法，即使在极高的拷贝数下也能准确测量串联重复序列，并将该技术应用于来自三个不同人群和五种非人灵长类动物的基因型165 HapMap样本。我们观察到串联重复基因拷贝数的极端变异性，许多基因座在人类中的拷贝数变异为5-10倍。这些基因座中的许多显示出基因组组装错误的标志，并且许多大串联重复序列的真实拷贝数即使在高质量的“成品”人类参考组装中也明显不足。重要的是，我们证明了大多数大的串联重复序列变异不是由附近的SNP标记的，因此基本上是不可见的基于SNP的GWAS方法。使用关联分析，我们确定了许多顺式相关的大串联重复序列变异与附近的基因表达和DNA甲基化水平，表明串联重复序列长度的变化与当地基因组环境的功能影响。这包括一个例子，其中一个大卫星重复的扩展与增加的DNA甲基化和附近的基因表达的抑制有关，这表明了一种称为“重复诱导的基因沉默”的机制，以前只在转基因生物中观察到。我们还观察到多个签名一致的改变选择压力在串联重复位点，这表明重要的生物功能。我们的研究表明，串联重复位点代表了基因组的一个高度可变的部分，已被系统地忽略了大多数以前的研究，拷贝数的变化，可以发挥功能显着的影响。我们认为，未来的研究串联重复基因座将导致许多新的见解，他们在调节基因组和表型多样性的作用。在这里，我们利用Nanostring数字分析，并显示其效用估计186多拷贝基因和串联重复的拷贝数。通过分析这些变体周围的单核苷酸变异模式，我们发现绝大多数串联重复序列变异的拷贝数变异并没有被附近的SNP有效标记，因此专注于SNP的标准全基因组关联研究很少或根本没有提供有关此类变体的信息。通过比较串联重复序列拷贝数与局部基因表达和DNA甲基化变化的模式，我们还确定了对局部基因组功能的广泛功能影响。这包括一个非编码宏卫星重复序列的例子，它的扩增对附近的基因产生抑制作用，伴随着局部DNA甲基化的积累。最后，不同的人类种群与一些灵长类动物基因组的比较表明，许多这些序列在最近的人类和灵长类动物进化过程中经历了极端的拷贝数变化，并显示出可能的选择性效应的签名。总体而言，我们的结论是，多拷贝基因和宏卫星代表了基因组的高度可变的一部分，具有重要的功能影响，已被系统地忽略了以前的研究。
Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity. Here we utilize Nanostring digital assays and show their utility for estimating copy number of 186 multicopy genes and tandem repeats. By analyzing patterns of single nucleotide variation around these variants, we show that copy number variation at the vast majority of tandem repeat variations is not effectively tagged by nearby SNPs, and thus standard genome-wide association studies that focus on SNPs provide little or no information about such variants. By comparing patterns of tandem repeat copy number with variation in local gene expression and DNA methylation, we also identify extensive functional effects on local genome function. This includes an example of a non-coding macrosatellite repeat, expansion of which exerts a repressive effect on a nearby gene accompanied by accumulations of local DNA methylation. Finally, comparison of diverse human populations with a number of primate genomes shows that many of these sequences have undergone extreme changes in copy number during recent human and primate evolution, and show signatures that suggest possible selective effects. Overall, we conclude that multicopy genes and macrosatellites represent a highly variable fraction of the genome with important functional effects that has been systematically ignored by previous studies.
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