Studies Of New Techniques For Nucleic Acid Test Using Next Generation Sequencing Platforms

The targets of nucleic acid test techniques are deoxyribonucleic acid(DNA)and ribonucleic acid(RNA).Uses of DNA detection include DNA sequence analysis,single nucleotide polymorphism(SNP)genotyping,copy number variation(CNV)detection,and DNA methylation detection.Uses of RNA detection include gene expression analysis and RNA sequence analysis.Nucleic acid test techniques have become indispensable in numerous applied fields such as basic biology,environmental science,and medical diagnosis.In the past decade,next generation sequencing(NGS)has developed rapidly,and has become one of the most important platforms for nucleic acid detection because of high-throughput and low cost.This study aims to make full use of the advantages of NGS,such as low cost,rapidity,accuracy and high throughput,to develop new nucleic acid detection techniques and extend their application for SNP genotyping,target regions capture and sequencing,and copy number variation detection.The following are the three main findings.First,study of SNP genotyping technique.This study used flexible,specific and repeatable multiplex PCR in combination with high-throughput NGS platforms to develop a SNP genotyping technique.There are two major challenges for traditional multiplex PCR coupled with NGS.The first challenge lies in the difficulty to have uniform abundance of amplicons within the sample.If there is a large amplification bias at different SNP loci,the products of the locus with high amplification efficiency will far exceed the products of the locus with low amplification efficiency.In the subsequent sequencing process,products of the locus with high amplification efficiency will occupy most of the sequencing throughput,resulting in insufficient sequencing depth of products of the locus with low amplification efficiency for accurate SNP genotyping.The second challenge is to ensure that samples with different concentrations have the similar amount of product.This variation among the samples could cause sequence coverage bias and reduce the sequencing efficiency.In this study,low concentrations of specific primers were used as restriction for PCR products.The equally low concentration primers were consumed as much as possible by a three-round PCR to ensure the uniformity of different loci and samples.Specifically,low concentration of specific primers were used for a few cycles in the first round PCR.In the second round PCR,part of products from the first round were added in an new PCR reaction system without adding primers,in order to consume remaining free residual primers from the first round.In the third round PCR,adapter primers were used to complete the library construction.In this study,37 loci from 757 human genomic DNA were amplified and all the amplicons were sequenced in a single run on an Ion Torrent PGM platform.Sequencing data showed that 98.6% of amplicons were successfully captured and sequenced,and 90.4% of amplicons had reads within a 50-fold abundance range.The high uniformity of amplicons ensured the efficiency of NGS.98.4% of samples reads fell within a 30-fold range.The method has good uniformity and is suitable for genetic analysis of large samples.Meanwhile,SNP genotyping thresholds based on NGS data was studied.Low sequencing cost and high sequencing throughput are the great advantages of NGS for SNP genotyping.However,with massive sequencing data,accurate SNP genotyping is still a challenge.Previous research experience has suggested that when the sequencing depth is >20 X,the loci with allele frequency in the range of 20% to 80% are heterozygous and the loci outside the range are homozygous.This threshold is accurate,simple,and intuitive.However,this threshold will filter out low sequencing depth loci(<20 X),resulting in a waste of sequencing throughput.At the same time,it is difficult to determine the loci around the allele ratio threshold by this method.The other SNP genotyping methods,such as low sequencing depths incorporated with linkage disequilibrium information used in the the 1000 Genomes Project or probabilistic methods,are not suitable for three-round multiplex PCR coupled with second-generation sequencing for SNP genotyping.Therefore,an accurate and appropriate SNP genotyping threshold is an important complement to SNP genotyping of large-scale samples with NGS.In this study,19 SNP loci from 91 samples were detected by ligase detection reaction(LDR),and their genotyping results were compared with the corresponding NGS data to determine the empirical cut-off threshold of NGS data for SNP calling: sequencing depth was ?6 X and heterozygote ratio was fell in 15%~85%.Application of this method was able to accurately determine 99.6% of SNPs but was failed to judge the data closed edge of the cut-off thresholds.Combining with clustering analysis could solve this problem for increasing the accuracy to 100%.The results of this research provided an accurate,fast and empirical threshold for NGS for single nucleotide polymorphism calling.Second,targeted enrichment using the blunt hairpin primers.Targeted enrichment sequencing is one of the main applications of NGS.The purpose of this study was to develop a target-enrichment technique using multiplex PCR by optimizing primer design.There is a key technical issue that needs to be solved.Multiplex PCR has been restricted by the dramatic increase in mispriming events and primer dimers as more primer pairs are used.These primer dimers and non-specific amplifications lead to less efficiency and bad uniformity of specific amplification.In this study,the blunt hairpin primer design for multiplex PCR was presented to improve primer specificity.Specifically,the 5'-end sequences of the hairpin primer was complementary about 13 bp to the 3'-end sequences.When the 3'-specific target sequences of the hairpin primer were identical to the template sequences about 20 bp,the hairpin primer annealed and hybridized with template to amplify.Otherwise,the hairpin primer annealed by itself and completely lock the 3'-end of primer.Compared with linear primers,the hairpin primers effectively reduced the nonspecific amplifications and primer dimers.89 target regions from 44 samples were simultaneously amplified and all amplicons were sequenced on an Ion Torrent PGM platform.Sequencing results showed that 95.1% of amplicons had reads within a 50-fold abundance range,and uniformity was excellent and similar to other reported multiplex PCR techniques.The results showed that multiplex PCR combined with the blunt hairpin primers was suitable for large-scale target-region capture sequencing.Third,extension application of the blunt hairpin primers for copy number variation detection.CNV has been recognized as an underlying factor for specific gene function and human disease,and therefore CNV research has great scientific value and clinical significance.Owing to the low cost and high repeatability,multiplex fluorescent competitive PCR(MFC-PCR)is an alternative method for CNV detection,but this method must break through two key technical issues.The first problem is to establish an efficient and stable multiplex PCR reaction system for reducing the influence of primer dimer and non-specific amplification.The second problem is how to construct a series of internal competitive templates economically,reliably,and conveniently,instead of traditional internal competitive constructed by artificial synthesis.In this study,the novel blunt hairpin primers used in targeted sequencing were extended in MFC-PCR to establish a novel copy number detection technique.The blunt hairpin primers were used to establishe efficient and stable multiplex PCR system.Simultaneously,the multiplex PCR were used for a few cycles with blunt hairpin primers,and constructed the internal competitive template instead of the traditional plasmid construction or artificial synthesis to avoid the complicated experimental operation and reagent cost.Specifically,the test sample and internal competitive sample were respectively amplified five cycles with the blunt hairpin primers in the first round of PCR,and the products of the test sample and the internal competitive sample are defined as the test template and the internal competitive template,respectively.The forward primers of the test sample had four key bases(ACTG)more than the primers of internal competitive sample to separate the PCR products of the test sample and internal competitive sample.The test template and the internal competitive template were mixed in equal amount and purified.In the second round of PCR,the mixed and purified products of the first round PCR were amplified using universal fluorescent primers,and the products of the universal fluorescent primers were directly detected by capillary electrophoresis.The PCR products of the test sample and internal competitive sample were identified by their fragment sizes.Then the fluorescence peak ratios of the test template to internal competitive template were calculated.Based on the principle of competitive PCR,CNV detection of multiplex fluorescence-competitive PCR is achieved by co-amplification of the test template and the internal sample with known copy number.By comparing the ratio of fluorescence between the test sample and the internal competitive template whose the copy number is known,you can calculate the number of copies of the test sample.In this study,21 samples with potential gene deletions were tested,and aCGH was used to evaluate the accuracy of MFC-PCR.The results of aCGH were completely consistent with the MFC-PCR results.The results showed that the extension application of the blunt hairpin primers for CNV detection is a cheap,efficient and convenient competitive PCR method and suitable for CNV detection of multiplex loci.