The Eastablishment And Application Of Multiple Competitive PCR Based On NGS Method

In systems genetics,analysis of deferentially expressed genes is an effective approach to provide abundant information about signal transduction,genes transcription,regulation and interaction as well as their spatio temporal expression patterns.The common techniques of gene expression analysis contain traditional competitive PCR,real-time quantitative PCR,micro array assay and other quantification methods derived from next generation sequencing(NGS)platforms.Selection of different methods is a case by case task because of their own characters.In this study,we combined multiple competitive PCR with Ion proton NGS platform together,to develop a novel gene expression quantification technique,particularly suitable for massive samples and genes of interest testing.The following application in systems genetics research of mouse(chromosome 1 substitution lines derived from Chinese wild mice)blood lipid demonstrate the feasibility of our new method.The key points of multiple competitive PCR based on NGS are: 1)construction of competitive templates.Single base mutation is introduced during reverse primer synthesis,thus,different competitors can be produced by reverse transcription;2)gradient-based mixture of competitors.Standard templates and target templates are mixed with a series of concentration ratio.Then,amplification efficiency can be calibrated by standard curve;3)uniformity of multiple PCR amplicons.Repeated adjustment of specific PCR primers ratio followed by real-time PCR.Quantification primers consist of a universal forward primer and a specific reverse primer;4)Ion proton sequencing.We wrote a whole pipeline for reads mapping,competitors identification and reads count,which can finish 10 G data analysis within several minutes.8 genes were chosen to evaluate the feasibility of this method.Our results show: 1)all the goodness of fit R2 > 0.98 demonstrate a better linear relationship than that of real-time PCR(0.97-0.99);2)the standard deviation of three experimental replications suggest good stability of both our method and real-time PCR(SD < 0.05);3)a good concordance(R2 > 0.98)between the two approaches with the ability to measure 2-fold variance.Totally,172 mice samples and 113 genes were tested using our technique and we found significant variance in gene expression among different lines,which suggest a potential relationship between transcripts abundance and blood lipid level.Meanwhile,more than 90%amplicons show a good uniformity within 30-fold change.Therefore,we not only solved over-sequencing and low-abundance transcripts sequencing problems but also supplied rich gene expression data for our future systems genetics research.In conclusion,we developed a novel gene expression analysis method via multiple competitive PCR and NGS technique.The stability,accuracy and sensibility were compared withthose of real-time PCR.Besides,application in systems genetics study of mouse blood lipid demonstrated a good uniformity of amplicons which can reduce the cost of sequencing.All the results implied our method have a good prospect in systems genetics research of complex diseases.