Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq
Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq
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DOI：
10.1038/s41596-020-0301-0
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发表时间：
2020-04-03
期刊：
影响因子：
14.8
通讯作者：
Vanderschuren, Herve
中科院分区：
文献类型：
--
作者：
Mehta, Devang;Cornet, Luc;Vanderschuren, Herve
环状DNA在自然界中以质粒、环状DNA病毒和真核生物中的染色体外环状DNA（eccDNA）的形式普遍存在。这些分子的测序对于分析病毒分布、发现新病毒和理解eccDNA在真核细胞中的作用是必不可少的。环状DNA富集测序（CIDER-Seq）是一种富集环状DNA并对其进行准确测序的技术，无需聚合酶链反应扩增、克隆和计算序列组装。该方法基于随机引物环状DNA扩增，随后是几个酶促DNA修复步骤，然后是长读序测序。CIDER-Seq包括一个定制的数据分析软件包（CIDER-Seq数据分析软件2），它实现了DeConcat算法，将随机环状DNA扩增的长测序产物解链为输入环状DNA的完整序列。CIDER-Seq数据分析包可以生成全长注释的病毒基因组，以及新型病毒的环状DNA序列。CIDER-Seq的应用还包括分析eccDNA分子，例如来自生物样品的转座因子（TE）。该方法需要2周才能完成，具体取决于可用的计算资源。由于目前长读单分子测序的限制，通过CIDER-Seq方法产生的环状病毒和eccDNA序列的准确度与序列长度成比例，并且对于分子获得最大的准确度。
Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes 2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules