A molecular method to determine isoform frequencies in RNA-seq

 说明书(由申请人提供)：当前的RNA-seq文库制备方法试图均匀地对每个mRNA分子上的所有序列进行采样，最佳地具有足够的重叠，以允许从其衍生的mRNA序列从头开始重组，或者可替换地，允许通过与参考序列的比对来推断mRNA序列。以多种异构体编码mRNAs的基因是一个挑战：给出一套完整的跨越每个外显子和剪接连接的短序列读取，某些替代的潜在mRNA异构体模型不能使用这种性质的数据来解卷。当一个以上的异构体模型可以解释外显子和连接序列阅读的频率时，就会出现这种混淆的情况，这在数学上是不可避免的：最终，短序列阅读不包含为某些常见剪接模式明确识别正确的异构体模型所需的信息。我们建议测试一种保存必要信息的方法。在这种方法中，目标是将单个条形码与来自同一mRNA分子的多个序列读取相关联，以及将不同条形码与从同一基因转录的彼此的mRNA分子的序列读取相关联。这将通过使用条形码随机引物随机引发合成cDNA来实现，在随机引发反转录过程中，每个mRNA分子暴露于一个且仅有一个条形码。原则上，这种方法产生分子特定的条形码cDNA集合，在高通量测序后，可以对其进行比对，以揭示逐个分子的mRNA异构体的特定结构细节。该方法解决了同构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.