The Construction And Application Study Of Multiplex Genotyping System With STR Loci For DNA Mixture Analysis By High-throughput Sequencing

Objective:Currently there exist many methods and the derived softwareanalysis systems about DNA mixture ananlysis and interpretation mainlybased on capillary electrophoresis(CE)-based separation technique forvariable-length classification of short tandem repeats(STRs). However, theprofile of this mainstream STR genotying technology which cannot generatethe whole STR sequence information have been interpratated hard withcompound repeats or complex repeats. On the other hand, developing-rapidlyNext-generation sequencing(NGS) not only generate allelic reads withhigh-coverage deep sequencing which provide the high probability inaccurately calling alleles at STR loci, but also achieve allelic nucleotidevariation information single nucleotide polymorphism(SNPs) that make theinverse sulotion of mixture probability models easier and more efficient withincreasing the key elements in the analysis of mixed DNA.Aimed at constructing a method of multiplex PCR genotyping system forlonger fragments(1000~1500bp) combination with a higher resolution ratio(1%) effective separating tecnology, the target STR loci PCR products rangefrom 900 bp to 1500 bp appropriatly with primers of 15 autosome STR inAmp FLSTR® Identifiler® system designed by the tools of NCBI-primer-blast,primer premier5.0, Oligo7 in the charge of products in 1:4 concentration,followed by fragment ananlysis(FA) detecting the length of fragmentsintuitively and exactly. Two of multiplex PCR systems are constructed withthe 15 STR loci divided into two groups for effective analysis of mixed DNAby next-generation sequencing platform, followed by primary evaluation tothe multiplex geotyping of mixture analysis.Methods: ①The primers of 15 autosome STRs loci with the sufficientpower of discrimination in Amp FLSTR® Identifiler® system(D8S1179,D21S11, D7S820, CSF1 PO, D3S1358, TH01, D13S317, D16S539, D2S1338,D19S433, VWA, TPOX, D18S51, D5S818, FGA) are designed by theprimer-designed software primer premier5.0, Oligo7 and the primer-designedwebsite http://www.ncbi.nlm.nih.gov/tools/primer-blast/, according to thereference sequence published by the website of http://www.ncbi.nlm.nih.gov/nuccore with the target STR loci PCR products ranging from 900 bp to 1500 bpappropriately and differences from 18 bp to 104 bp. ②Construction of STRsmultiplex PCR reaction system: the two groups of multiplex PCR productsfragments are separated and detected with high resolution by FragmentAnalyzer? Automated CE System(AATI) respectively after adjustmentprimers and concentration with Platinum® Multiplex PCR Master Mix(ABI),followed by the data collection and analysis in PROSize® data analysissoftware in the charge of products in 1:4 concentration. ③The mixtureresearch model constructed by known STR genotyping female(9947A) andmale(2800, 007) control DNAs(promega) are amplified by this STRsmultiplex genotyping system, subsequently the specificity of this systemprimers and the application value of this multiplex PCR system in mixtureanalysis are confirmated and evaluated by Illumina® Mi Seq® System.Results:1 The construction of 15 autosome STRs loci multiplex genotypingsystemThe multiplex genotyping system multiplex genotyping system isconstructed with the 15 STR loci divided into two groups-one is 8 loci and theother is 7 loci ranged from 900 bp to 1500 bp. The results from FA showed that,every locus fragment(s) in DNA mixture could be mapped that in singlesample within 1:4 fragments concentration, without non-specificity fragments.2 The specificity of this system primers and the application value of thismultiplex PCR system in mixture analysis are confirmated and evaluated byIllumina® Mi Seq® SystemThe sequencing results showed that this multiplex PCR system couldamplify well 15 autosome STR loci in sigle sample without non-specificityamplicon in mapping human genome. On the other side,sequencing results inmixed DNA showed that regardless of the sttuers besides major componentbeing merged or eliminated, the alleles in locus were in accordance with theexpected distribution with allelic imbalance(Alm) in some loci and one allelicdrop-out.Conclusions:1 By the way of designing PCR primers, two parallel multiplex PCRgenotyping systems are successfully developed, which including 15 autosomeSTR loci with the sufficient power of discrimination and the productfragments about 1000 bp, and it would serve an important role for analyzingchallenging forensic mixture samples with next-generation sequencing.2 This multiplex PCR system of 15 autosome STR loci was constructedsuccessfully for detecting genotyping by next-generation sequencing fromsingle sample,what’s more sequencing results of mixed DNA through thismultiplex PCR system amplification could be analysed in genotypingidentification,but still need to be further optimizated and improved.