BACKGROUND Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even "third generation" sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly. RESULTS We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall. CONCLUSIONS This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation.

中文翻译：

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在单次测序读取中捕获完整的纤毛虫染色体揭示了广泛的染色体亚型。

背景技术全基因组鸟枪测序将数百万个短测序读段拼接成单个基因组，开创了现代基因组学时代，并导致可用基因组序列数量的快速扩展。然而，短读长的组装仍然很困难，导致基因组序列碎片化。最终，只有能够在一次运行中捕获完整染色体的测序技术才能解决所有歧义。即使是“第三代”测序技术产生的读数也比大多数真核染色体短得多。然而，纤毛虫 Oxytricha trifallax 的体细胞基因组包含数千条染色体，平均长度仅为 3.2 kbp，使其成为探索无需组装即可对整个染色体进行测序的优势的理想候选者。结果我们使用单分子实时测序在单次读取中捕获数千条完整染色体，并更新已发表的三毛尖毛虫 JRB310 基因组组装。在此版本中，超过 50% 的具有两个端粒的完整染色体来自单次读取。改进的组装包括超过 12,000 个新的染色体亚型，并证明体细胞染色体源自编码多达 191,000 个碱基对的体细胞片段之间的可变重排。然而，虽然长读长减少了组装的需要，但用短读长进行纠错来补充长读长测序的混合方法总体上产生了最完整、最准确的组装。结论 该组装提供了通过单次测序读取捕获完整真核染色体的第一个例子，并证明传统的基因组组装方法可以掩盖相当大的结构变异。