We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone.

中文翻译：

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部分亚硫酸氢盐转化可实现独特的模板测序

我们引入了一种新的协议，即突变测序或muSeq，它使用亚硫酸氢钠以固定且可调的速率将氨基甲基化的胞嘧啶随机脱氨基。muSeq协议使用模板的每个副本以及副本的每个片段副本中存在的唯一突变特征标记每个初始模板分子。在测序的读取数据中，此签名被视为C到T或G到A核苷酸转换的独特模式。具有相同转换模式的聚类读取可从短读取序列数据中对初始模板分子进行精确计数和长距离组装。我们通过在PstI表示中分析135 000个限制性片段来研究计数和低错误测序，证明muSeq可以改善拷贝数推断并显着降低偶发性测序错误。我们探索在cDNA的背景下的远程组装，生成长度大于3,000 bp的连续转录簇。muSeq程序集揭示了仅从短读数据中无法观察到的转录多样性。