A method to determine mRNA isoform frequencies using novel primer generators

摘要 目前用于RNA-seq文库制备的方法试图对所有序列进行均匀取样。 每个mRNA分子，最好有足够的重叠，以允许重新组装的mRNA序列 或可替代地，允许通过与参考序列比对来推断mRNA序列 序列的以多种亚型编码mRNA的基因提出了一个挑战：给定一套完整的短 跨越每个外显子和剪接点的序列读段，某些替代的潜在mRNA亚型模型 不能使用这种性质的数据来解卷积。这种混淆情况发生在多个 异构体模型可以解释外显子和连接序列读数的频率，并且在数学上是 不可避免的：最终，短序列读段不包含明确识别 某些常见剪接模式的正确同种型模型。我们建议测试一种方法，以保持 信息来重建异构体模型。在这种方法中，我们生成一个小的条形码集合， 每个单独的mRNA分子的重叠序列读段，使得来自相同mRNA分子的序列读段 mRNA分子含有相同的条形码，但来自同一基因的其他转录物各自与 一个不同的分子特异性条形码重叠群的组装需要基因衍生的序列的比对。 与相同条形码相关的序列。这将通过随机引物合成的cDNA在一个 使用珠粒的乳液形式，每个珠粒携带随机引物，所述随机引物侧接珠粒特异性条形码。一 该提议的新奇是产生其中每个珠仅携带一个条形码的珠文库的方法， 但是其中条形码的总体复杂性非常高。这些珠子用于生成条形码 也含有cDNA的乳液液滴中的随机引物，使得多个随机引发的产物都 都有相同的条形码原则上，该方法产生分子特异性的条形码化的多肽集合。 cDNA，其在高通量测序时可以被比对以揭示DNA的特定结构细节。 mRNA同种型在分子-分子基础上。这种方法将解决异构体模型的可识别性 问题. 1

Abstract 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 information to reconstruct isoform models. In this method, we generate a small barcoded collection of overlapping sequence reads for every individual mRNA molecule, such that sequence reads from the same mRNA molecule contain the same barcode, but other transcripts from the same gene are each associated with a different molecule-specific barcode. Assembly of contigs entails the alignment of the gene-derived sequences associated with the same barcode. This will be done by random primed synthesis of cDNA in an emulsion format using beads each of which carries random primers flanked by a bead-specific barcode. A novelty in this proposal is a method to generate a bead library in which each bead carries only one barcode, but in which the overall complexity of barcodes is very high. These beads are used to generate barcoded random primers in emulsion droplets that also contain cDNA, such that multiple randomly primed products all contain the same barcode. 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. 1