基于本地BLAST的高通量引物设计R包(LightPrimer)及其应用

A local BLAST based high throughput primer designing R package(LightPrimer)and its application

引物优劣是PCR反应成败的重要影响因素之一,当前引物设计软件存在通量低、操作复杂、开源性不高等不足。本文开发了一款基于R语言和本地BLAST的针对克隆、qRT-PCR、SNP和InDel标记的高通量引物设计软件(LightPrimer)。该软件通过序列信息从基因组当中提取特定序列,进行片段化处理,然后将片段与基因组进行本地BLAST比对,通过序列特异性指数和比对位点数筛选高特异性片段,然后对Tm值、GC含量、扩增片段长度、引物长度、引物3’末端匹配、末端GC碱基和引物二聚体进行筛选,得到候选引物列表,并通过序列评价诊断图为引物优化提供参考。该软件具有高通量、操作简便、跨平台、开源等特点,可为现有引物设计软件提供有益补充。软件可从github下载(https://github.com/YangtzeSoyGDB/LightPrimer.git)。

The primer quality is one of the important factors affecting PCR reaction.The shortcomings of current primer design software mainly lie in relatively low throughput,complicated operation process and less extent of source opening.In this study,a high-throughput primer designing software(LightPrimer)was developed for cloning,quantitative real time PCR(qRT-PCR),SNP and In Del markers development based on R and Local-BLAST.LightPrimer extracts specific sequences from the working genome via sequence information,followed by sequence fragmentation,Then basic local alignment between fragmentated sequences and the genome is performed by Local-BLAST,screening of highly specific fragmentated sequences which are filtrated through sequence specificity index and matched loci.After that a list of candidate primers is to be obtained by filtration of Tm,GC content,amplicon length,primer length,3’end matching,GC end base and dimer screening.The sequence evaluation diagnostic plot could provide a reference for primer optimization if no target primers were obtained.LightPrimer is of high throughput,simple operation process,cross platform and open source,which could be a useful supplement to the existing primer design software.The LightPrimer can be downloaded from git Hub(https://github.com/YangtzeSoyGDB/LightPrimer.git).