MP3RNA-seq:Massively parallel 3’end RNA sequencing for high-throughput gene expression profiling and genotyping

摘要

Transcriptome deep sequencing(RNA‐seq)hasbecome a routine method for global geneexpression profiling.However,its application tolarge‐scale experiments remains limited by costand labor constraints.Here we describe amassively parallel 3′end RNA‐seq(MP3RNA‐seq)method that introduces unique samplebarcodes during reverse transcription to permitsample pooling immediately following this initialstep.MP3RNA‐seq allows for handling of hun-dreds of samples in a single experiment,at acost of about$6 per sample for libraryconstruction and sequencing.MP3RNA‐seq iseffective for not only high‐throughput geneexpression profiling,but also genotyping.Todemonstrate its utility,we applied MP3RNA‐seqto 477 double haploid lines of maize.We iden-tified 19,429 genes expressed in at least 50%ofthe lines and 35,836 high‐quality singlenucleotide polymorphisms for genotypinganalysis.Armed with these data,we performedexpression and agronomic trait quantitativetrait locus(QTL)mapping and identified 25,797expression QTLs for 15,335 genes and 21 QTLsfor plant height,ear height,and relative earheight.We conclude that MP3RNA‐seq is highlyreproducible,accurate,and sensitive forhigh‐throughput gene expression profiling andgenotyping,and should be generally applicableto most eukaryotic species.