A method to determine mRNA isoform frequencies using novel primer generators

AbstractCurrent methods for RNA seq library preparation attempt to uniformly sample all sequences across every mRNA moleculeoptimally with sufficient overlap to allow de novo reassembly of the mRNA sequences from which they deriveor alternativelyto allow inference of mRNA sequence by alignment with reference sequencesGenes that encode mRNAs in multiple isoforms present a challengegiven a complete set of short sequence reads that span every exon and splice junctioncertain alternative underlying mRNA isoform models cannot be deconvoluted using data of this natureThis confounding situation occurs when more than one isoform model can explain the frequencies of exon and junction sequence readsand it is mathematically unavoidableultimatelyshort sequence reads do not contain the information needed to unambiguously identify the correct isoform model for certain common splicing patternsWe propose to test a method to preserve the information to reconstruct isoform modelsIn this methodwe generate a small barcoded collection of overlapping sequence reads for every individual mRNA moleculesuch that sequence reads from the same mRNA molecule contain the same barcodebut other transcripts from the same gene are each associated with a different molecule specific barcodeAssembly of contigs entails the alignment of the gene derived sequences associated with the same barcodeThis 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 barcodeA novelty in this proposal is a method to generate a bead library in which each bead carries only one barcodebut in which the overall complexity of barcodes is very highThese beads are used to generate barcoded random primers in emulsion droplets that also contain cDNAsuch that multiple randomly primed products all contain the same barcodeIn principlethis method produces molecule specific collections of barcoded cDNAswhichupon high throughput sequencingcan be aligned to reveal the specific structural details of mRNA isoforms on a molecule by molecule basisThis approach would solve the isoform model identifiability problemProject Narrative High throughput sequencing methods that generate short sequence reads cannot be used to reliably describe or quantify the different mRNA isoforms that can arise due to alternative splicing from genes that haveor more exonsThe method we propose solves this problem by barcoding each subsequence from a single mRNA with exactly the same unique barcode