A method to determine mRNA isoform frequencies using novel primer generators

使用新型引物生成器确定 mRNA 同工型频率的方法

• 批准号: 9622969
• 负责人: JOHN T WELSH
• 金额: $ 22.5万
• 依托单位: VLP BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9622969
• 关键词: Alternative Splicing; Biological Process; Biology; Cells; Collection; Complementary DNA; Complex; Data; Data Analyses; Development; Emulsions; Exons; Frequencies; Gene Expression; Genes; Goals; High-Throughput Nucleotide Sequencing; Human; In Vitro; Individual; Length; Libraries; Mathematics; Medicine; Messenger RNA; Methods; Modeling; Molecular Biology; Nature; Oligonucleotides; Pathology; Pattern; Phenotype; Plant Resins; Polyadenylation; Population; Preparation; Primer Extension; Problem Solving; Protein Isoforms; Proteins; RNA; RNA Splicing; Recording of previous events; Sampling; Sequence Alignment; Spliced Genes; Structure; Testing; Time; Tissue-Specific Gene Expression; Transcript; base; experimental study; instrument; mRNA Stability; novel; novel strategies; promoter; statistics; transcriptome; transcriptome sequencing

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

抽象的 当前的 RNA-seq 文库制备方法尝试对跨区域的所有序列进行统一采样 每个 mRNA 分子，最好有足够的重叠，以允许 mRNA 序列从头重新组装 它们从中衍生，或者允许通过与参考比对来推断 mRNA 序列 序列。编码多种亚型 mRNA 的基因提出了一个挑战：给定一整套短序列 跨越每个外显子和剪接点的序列读取，某些替代的底层 mRNA 同种型模型 无法使用这种性质的数据进行解卷积。当超过一个时，就会出现这种令人困惑的情况 isoform模型可以解释外显子和连接序列读取的频率，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所需的信息 某些常见剪接模式的正确异构体模型。我们建议测试一种方法来保存 重建异构体模型的信息。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 分子的重叠序列读取，使得序列读取来自相同的 mRNA 分子含有相同的条形码，但来自同一基因的其他转录本各自与 不同的分子特异性条形码。重叠群的组装需要基因衍生的比对 与相同条形码相关的序列。这将通过随机引物合成 cDNA 来完成 使用珠子的乳液形式，每个珠子都带有随机引物，两侧是珠子特异性条形码。一个 该提案的新颖之处在于一种生成珠子库的方法，其中每个珠子仅携带一个条形码， 但其中条码的整体复杂度非常高。这些珠子用于生成条形码 乳液滴中的随机引物也含有 cDNA，因此多个随机引物都 包含相同的条形码。原则上，该方法产生条形码的分子特异性集合 cDNA，通过高通量测序，可以比对以揭示 cDNA 的具体结构细节 逐个分子基础上的 mRNA 同工型。这种方法将解决异构体模型的可识别性 问题。 1