A molecular method to determine isoform frequencies in RNA-seq

 描述（由申请人提供）：目前用于RNA-seq文库制备的方法试图对每个mRNA分子中的所有序列进行均匀取样，最佳地具有足够的重叠以允许重新组装它们所来源的mRNA序列，或者允许通过与参考序列比对来推断mRNA序列。以多种亚型编码mRNA的基因提出了一个挑战：给定一组完整的跨越每个外显子和剪接点的短序列读段，某些替代的潜在mRNA亚型模型不能使用这种性质的数据去卷积。当不止一种亚型模型可以解释外显子和连接序列读数的频率时，就会出现这种混淆情况，并且在数学上是不可避免的：最终，短序列读数不包含明确识别某些常见剪接模式的正确亚型模型所需的信息。我们建议测试一种方法来保存必要的信息。在这种方法中，目标是将单个条形码与来自相同mRNA分子的多个序列读数相关联，并将不同的条形码与来自相同基因转录的每个其他mRNA分子的序列读数相关联。这将通过使用条形码化随机引物的cDNA的随机引发合成来完成，其方式使得每个mRNA分子在随机引发逆转录期间暴露于一个且仅一个条形码。原则上，该方法产生分子特异性的条形码化cDNA集合，其在高通量测序时可以被比对以在分子-分子的基础上揭示mRNA同种型的特定结构细节。这种方法将解决异构体模型的可识别性问题。

 DESCRIPTION (provided by applicant): Current methods for RNA-seq library preparation attempt to uniformly sample all sequences across every mRNA molecule, optimally with sufficient overlap to allow de novo reassembly of the mRNA sequences from which they derive, or alternatively, to allow inference of mRNA sequence by alignment with reference sequences. Genes that encode mRNAs in multiple isoforms present a challenge: given a complete set of short sequence reads that span every exon and splice junction, certain alternative underlying mRNA isoform models cannot be deconvoluted using data of this nature. This confounding situation occurs when more than one isoform model can explain the frequencies of exon and junction sequence reads, and it is mathematically unavoidable: ultimately, short sequence reads do not contain the information needed to unambiguously identify the correct isoform model for certain common splicing patterns. We propose to test a method to preserve the necessary information. In this method, the goal is to associate a single barcode with multiple sequence reads from the same mRNA molecule, and different barcodes with sequence reads from each other mRNA molecule transcribed from the same gene. This will be done by random primed synthesis of cDNA using barcoded random primers in such a manner that each mRNA molecule is exposed to one, and only one, barcode during random primed reverse transcription. In principle, this method produces molecule-specific collections of barcoded cDNAs, which, upon high throughput sequencing, can be aligned to reveal the specific structural details of mRNA isoforms on a molecule-by-molecule basis. This approach would solve the isoform model identifiability problem.