Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approaches have surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from > 15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten commonly-used de novo assemblers, and occurs over a range of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

中文翻译：

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变异干扰对病毒深度测序从头组装的影响。

病毒具有很高的突变率，通常在生物样品中以变体的混合物形式存在。下一代测序（NGS）方法在产生长病毒序列方面已经超过了桑格（Sanger），但是，变体如何影响NGS从头组装仍然尚待探索。我们从15,000多个模拟实验中获得的结果表明，变异体的存在可以将一个基因组的组装转变为数十个至数千个重叠群。这种“变异干扰”（VI）具有高度的一致性，并且可以由十个常用的从头组装者重现，并且发生在基因组长度，读取长度和GC含量的范围内。VI的主要驱动力是病毒变体之间的成对身份。计算机模拟进一步支持了这些发现，从临床数据集中选择性删除次要变异读段，可以从片段化的重叠群中“拯救”整个病毒基因组。这些结果要求仔细解释病毒深度测序中从头组装产生的重叠群和重叠群编号。