Large insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries. A Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~ 8 kb) to be fragmented into 2–3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5–10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations. We developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

中文翻译：

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PacBio 对 Fosmid 文库进行高通量长配对末端测序

大型插入片段双末端测序技术是组装基因组、描绘相关断点和检测结构重排的重要工具。为了便于全面检测染色体间和染色体内结构重排或变异（SV）以及具有长重复和片段重复的复杂基因组组装，我们开发了一种基于单分子实时合成测序技术的新方法，用于生成长配对大型插入 DNA 文库的末端序列。Fosmid 载体 pHZAUFOS3 具有以下新特征：（1）两个 18 bp 非回文 I-SceI 位点位于克隆位点的两侧，另外两个位点存在于载体骨架中，允许长 DNA 插入片段（以及本文中的长配对末端）作为单个片段回收，载体（~8 kb）通过 I-SceI 消化被分成 2-3 kb 片段，因此被有效地从长双端（5-10 kb）；(2)克隆生长所必需的氯霉素(Cm)抗性基因和复制子(oriV)位于克隆位点的两侧附近，有助于增加双端片段与单端片段的比例。双端库。通过将大小选择、机械剪切的混合 Fosmid DNA 片段连接到氨苄青霉素 (Amp) 抗性基因片段并用 Cm 和 Amp 筛选菌落来构建双末端文库。我们在酵母和狗尾草 Yugu1 上测试了这种方法。生成了平均长度超过 2 kb 的 Fosmid 大小的双端。酵母和 S. italica Yugu1 基因组从头组装的 N50 支架长度显着提高。在 S. italica Yugu1 中鉴定出五个大和五个小的结构重排或组装错误，跨越几十个 bp 到几十个 kb，包括缺失、倒位、重复和易位。我们开发了一种对大型插入文库进行长配对末端测序的新方法，该方法可以有效提高从头基因组组装的质量，并识别大小结构重排或组装错误。在 S. italica Yugu1 中鉴定出五个大和五个小的结构重排或组装错误，跨越几十个 bp 到几十个 kb，包括缺失、倒位、重复和易位。我们开发了一种对大型插入文库进行长配对末端测序的新方法，该方法可以有效提高从头基因组组装的质量，并识别大小结构重排或组装错误。在 S. italica Yugu1 中鉴定出五个大和五个小的结构重排或组装错误，跨越几十个 bp 到几十个 kb，包括缺失、倒位、重复和易位。我们开发了一种对大型插入文库进行长配对末端测序的新方法，该方法可以有效提高从头基因组组装的质量，并识别大小结构重排或组装错误。