| Background: With the advancement of technology and the reduction of prices in recent years,high-throughput sequencing technology has become an important method in the filed of biology.Targeted Sequencing is a technique aimed at capturing specific regions of genome followed by sequencing with high depth.It can be used in precision medicine,population genetics,cancer sequencing,QTL candidate gene re-sequencing,mitochnodria resequencing,molecular breeding background selection,genetic disease detection and other aspects.Especially in clinical practice,target sequencing for dentifying genetic disease mutations and tumor mutations become more popular.Compared to Whole-Genome or Whole-Exons Sequencing,targeted sequencing has the advantage of using a rapid enrichment method based on multiplex PCR or oligonucleotide probe hybridization to capture only interested DNA for sequencing,resulting in increasing sequencing depth.Moreover,it can reduce the cost of sequqncing and shortens the research time.A key point of multiplex PCR technology is to design a rational multiplex PCR primer pair combinations,to ensure that there are no overlapping amplicon in the primer combination,and to reduce the interaction among the primers.At present,though multiplex PCR primer design can be performed by primer design software,there are still some problem need to be solved.PrimerStation,MultiPLX,MPprimer and BatchPrimer3 are free to use,but the specific software for designing primers of Targeted Sequencing based on multiplex PCR are still unpresented.Thus,the design process of multiplex PCR target caputure primers is complex,time consuming and labor intensive,which should be accomplished with multiple software together.Therefore,a multiplex PCR primer design software for targeted sequencing needs to be developed.The main task of this article is to develop a multiplex PCR primer design software for targeted sequencing,which can be divided into three parts: Firstly,the UCSC native gene annotation(HG38 genome)database will be established to achieve local input of interested geneor region to obtain DNA directly,avoiding users to frequently use NCBI or Ensembl database to retrieve and download related sequences;Secondly,a primer design module based on primer3-py for multiple sequences was developed to cover all target regions;Finaly,a module was developed to evaluate the interaction between primers and to detect the formation of overlapping amplification regions between primes and to automate the grouping.1)Establish a local interested sequence acquisition moduleBioinformatics databases(NCBI,Ensembl,UCSC,etc.)are widely used awith public data downloading iservice.In current study,UCSC database was used to download the human reference genome sequence and the genes annotation information(including gene name,number of gene exons,chromosome where gene is located,exon start position and termination position,etc.).After downloading,the relational database software MySQL was used to build a database of loal gene annotaion information,integrated with gene annotation information and gene sequence files into entire module.This module is mainly used to receive the target gene of target location information input by users,and then converts the input information into DNA sequence data for primer design.2)Multiple sequence candidate primer design moduleAlthough there are many kinds of existing multiplex PCR primer design software,most of these software are developed based on primer3 for specific purposes.However,the operation of them are troublesome and time-consuming.Moreover,as the interested sequence increases,it also causes the programs to teminate prematurely because there is no suitable primer pairs set returned.Therefore,we developed a multiple sequence primer design module based on primer3 for multiplex PCR primers used for Targeted Sequencing.In searching for all candidate primer pairs in the interested sequence,the module mainly uses the primer self factor,duch as primer GC content,primer dissolving temperature Tm,length of primer,etc.PCR product length as a limiting condition,irrespective of the formation of a stable secondary structure between the primers.The primer design process between multiple interested sequences is independent.We used a multi-process approach to speed up the primer design process.In the process of designing mutiplex PCR primers for Targeted Sequencing,how to abtain all candidate primer pair sets for each interested region and determine whether there is a set of primer pairs covering the entire region,is one of key issues.Each interested region will be determined individually.First,all candidate primer pairs in the interested region are sorted according to the start position of the left primer,then the candidate primer pair group is traversed,and the area to be covered each time is updated until the entire interested region is covered.When there are areas that cannot covered,traversal is terminated early.3)The pre-amplification primer set are grouped according to the interaction and overlapping areas between the primersAt present,there are quite a few software for multiplex PCR primer analysis,but most of them only analyze the non-specfic amplification of new amplicons formed in the primer set,and formed stable secondary structures between the primers.There is not a grouping solution available.Although the primer grouping software MultiPLX can calculate the interaction between primers and achieve automatic grouping,the situation of overlapping amplicon bwtween primers was not considerred.In this study,we used the thermodynamic alignment module of primer3 to develop a grouping module base on the overlap between primers.In this module,the primers designed by the multi-sequence primer design module are first subjected to a secondary structure operation between the primers to generate an N × N two-dimensional table.The secondary structure between the primers is larger than the threshold(△G is less than-8 Kj/mol),penalties are given and grouped based on the non-overlapping and the penalty table between the pairs of primers.Conclusion: Our system can achieve the mixed input of the interested gene or region to design a multiplex PCR reaction sets.The designed primer pair set is mainly divided into two groups of multiplex PCR primer sets,and the primer pair combination can achieve the full coverage target area,and in each pre-amplification multiplex PCR primer set,there was minimal interaction between primers.Targeted Sequencing gene detection and SNP genotyping using a multiplex PCR capture protocol provides a good,available and complementary tool.Using this system,we designed a multiplex PCR targeted gene exon sequencing experiment scheme for BRCA1 and BRCA2 exons and verified the system.As a result,TASPrimer software designed 130 pairs of primers and automatically divided into two sets of multiplex PCR reactions based on the interaction and overlap between the primers.In the specific experimental results,the fluorescence quantification and Next Generation Sequencing results showed that each amplicon was amplified efficiently,demonstrating the availability and good assistance of the TASPrimer software in targeted analysis and sequencing experiments based on multiplex PCR technology. |
