A molecular method to determine isoform frequencies in RNA-seq

确定 RNA-seq 中异构体频率的分子方法

• 批准号: 8929900
• 负责人: JOHN T WELSH
• 金额: $ 26.95万
• 依托单位: VACCINE RESEARCH INSTITUTE OF SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8929900
• 关键词: Accounting; Alternative Splicing; Biological Process; Biology; Collection; Complementary DNA; Complex; DNA biosynthesis; Data; Exons; Frequencies; Gene Expression; Gene Expression Profile; Generations; Genes; Goals; High-Throughput Nucleotide Sequencing; In Vitro; Individual; Length; Libraries; Marketing; Measurement; Medicine; Messenger RNA; Methods; Modeling; Molecular; Molecular Biology; Nature; Oligonucleotides; Pattern; Plant Resins; Poly(A) Tail; Polyadenylation; Population; Preparation; Problem Solving; Protein Isoforms; Proteins; RNA; RNA Splicing; Reading; Recording of previous events; Reverse Transcription; Sampling; Sequence Alignment; Structure; Surface; Synthesis Chemistry; Testing; Time; Tissue-Specific Gene Expression; base; instrument; mRNA Stability; novel; oligo (dT); promoter; public health relevance; research study; statistics; transcriptome sequencing

 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.

 描述（由申请人提供）：当前的RNA-seq文库制备方法试图对每个mRNA分子的所有序列进行统一采样，最好有足够的重叠，以允许从头重新组装它们所衍生的mRNA序列，或者允许通过与参考序列比对来推断mRNA序列。编码多种同种型 mRNA 的基因提出了一个挑战：给定跨越每个外显子和剪接点的完整短序列读取集，某些替代的基础 mRNA 同种型模型无法使用这种性质的数据进行解卷积。当多个异构体模型可以解释外显子和连接序列读取的频率时，就会出现这种令人困惑的情况，并且在数学上是不可避免的：最终，短序列读取不包含明确识别某些常见剪接模式的正确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。在此方法中，目标是将单个条形码与来自同一 mRNA 分子的多个序列读数相关联，并将不同的条形码与来自同一基因转录的每个其他 mRNA 分子的序列读数相关联。这将通过使用带条形码的随机引物以随机引物合成 cDNA 的方式来完成，其中每个 mRNA 分子在随机引物逆转录过程中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 cDNA 集合，通过高通量测序，可以对这些集合进行比对，以逐个分子的方式揭示 mRNA 异构体的特定结构细节。这种方法将解决异构体模型的可识别性问题。