Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.     

MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays

We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5′ end followed by locus-specific sequence at the 3′ end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of ~95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of ~75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.     

In silico single nucleotide polymorphism discovery and application to marker-assisted selection in soybean

The general approach to discovering single nucleotide polymorphisms (SNPs) requires locus-specific PCR amplification. To enhance the efficiency of SNP discovery in soybean, we used in silico analysis prior to re-sequencing as it is both rapid and inexpensive. In silico analysis was performed to detect putative SNPs in expressed sequence tag (EST) contigs assembled using publicly available ESTs from 18 different soybean genotypes. SNP validation by direct sequencing of six soybean cultivars and a wild soybean genotype was performed with PCR primers designed from EST contigs aligned with at least 5 out of 18 soybean genotypes. The efficiency of SNP discovery among the confirmation genotypes was 81.2%. Furthermore, the efficiency of SNP discovery between Pureunkong and Jinpumkong 2 genotypes was 47.4%, a great improvement on our previous finding based on direct sequencing (22.3%). Using SNPs between Pureunkong and Jinpumkong 2 in EST contigs, which were linked to target traits, we were able to genotype 90 recombinant inbred lines by high-resolution melting (HRM) analysis. These SNPs were mapped onto the expected locations near quantitative trait loci for water-logging tolerance and seed pectin concentration. Thus, our protocol for HRM analysis can be applied successfully not only to genetic diversity studies, but also to marker-assisted selection (MAS). Our study suggests that a combination of in silico analysis and HRM can reduce the cost and labor involved in developing SNP markers and genotyping SNPs. The markers developed in this study can also easily be applied to MAS if the markers are associated with the target traits.     

一种基于适配器连接介导的等位基因特异性扩增法测定多重SNP

建立了一种基于DNA适配器连接介导的等位基因特异性扩增法测定多重SNP。以CYP2D6基因中的5个SNP位点（100C>T,1661G>C,1758G>T,2470T>C和2850C>T）为例,用PCR法预扩增得一段含所有待测SNP位点的长片段,然后用限制性内切酶将其消化成短片段,在连接酶的作用下与设计的DNA适配器（adapter）相连;该适配器的一端与限制性内切酶降解后留下的粘性末端相同,另一端带有一段公共序列。在两管中加入与适配器连接的片段作为PCR扩增模板,并分别加入SNP特异性引物和一种适配器特异性的通用引物进行PCR扩增,最后用凝胶电泳法分离PCR扩增产物。由于每管与SNP的两种特异性引物中的一种对应,可以根据每管中扩增片段的大小判断SNP的类型。通过凝胶电泳法可以一次分离与5种SNP类型相对应的引物特异性延伸反应产物;采用该法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与限制性片段长度多态性法（RFLP）测定结果完全一致。该方法采用n+1种引物（n种SNP特异性引物和一种通用引物）进行n重PCR反应,极大提高了PCR反应的特异性,结果准确,可用于同时测定多个SNP位点。

     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.     

一种优化的胡杨高效多重（12重）SSR体系

微卫星多重PCR方法是一种非常经济并且高通量的基因分型技术。本研究在耐干旱、盐碱的胡杨（Populus euphratica）中开发出一套荧光标记的12重微卫星工作体系。该体系包含12条表达序列标签微卫星（EST-SSR）引物,其中3条设计于NCBI,另外9条设计于二代的转录组序列。利用该多重微卫星体系可在单一的PCR反应体系中成功扩增出12条表达序列标签的微卫星短序列片段。并在胡杨的3个自然居群96个个体中对该体系进行了验证,结果显示该体系具有很高的稳定性及多样性。同时,在杨属的5个派7个种中对其通用性进行了检验,显示这些引物具有很高的通用性,成功扩增率为79％。本研究中提供的12重多重PCR结合本实验已经公开发表的2个8重体系对揭示胡杨及其他杨树的进化历史具有重要的作用。最后,本研究认为引物的选择,扩增效率,哑等位基因的检测是多重体系开发过程中最为关键的步骤。     

高通量飞行时间质谱基因分型方法的研究

为了考察飞行时间质谱基因分型方法 (MALDI-TOF) 的位点分型成功率和分型结果质量的关系,分析了 96 个 SNPs 位点的近 10 000 个基因分型数据 (用 MALDI-TOF “4 重”实验方法检测 ). 结果显示,位点分型成功率和分型结果的质量显著正相关 . 分型成功率低于 82% 的 SNP 位点,其高质量结果占的比例开始逐渐降低 . 提示 82% 的分型成功率可以作为衡量分型结果质量的数据点 . 为了进一步提高通量并降低成本,在 MALDI-TOF “ 4 重”实验方法的基础上,发展了两种“准 8 重”实验方法 . 用新的实验方法检测了 95 个样本的 32 个 SNPs 位点 . 结果显示“混合准 8 重”实验方法与“ 4 重”实验方法相比无显著差异,而“复点准 8 重”的结果差于“ 4 重”分型方法 .     

SNPWave: a flexible multiplexed SNP genotyping technology

Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.     

An innovative SNP genotyping method adapting to multiple platforms and throughputs

Key message

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility.

Abstract

Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3′ regions to significantly increase the amplification specificity of the two alleles and tailed at 5′ ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion–deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

     

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.     

SNPWaveTM: a flexible multiplexed SNP genotyping technology

Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave^TM, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP®) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.     

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

DNA sequencing has markedly changed the nature of biomedical research, identifying millions of polymorphisms along the human genome that now require further analysis to study the genetic basis of human diseases. Among the DNA-sequencing platforms available, Pyrosequencing has become a useful tool for medium-throughput single nucleotide polymorphism (SNP) genotyping, mutation detection, copy-number studies and DNA methylation analysis. Its 96-well genotyping format allows reliable results to be obtained at reasonable costs in a few minutes. However, a specific biotinylated primer is usually required for each SNP under study to allow the capture of single-stranded DNA template for the Pyrosequencing assay. Here, we present an alternative to the standard labeling of PCR products for analysis by Pyrosequencing that circumvents the requirement of specific biotinylated primers for each SNP of interest. This protocol uses a single biotinylated primer that is simultaneously incorporated into all M13-tagged PCR products during the amplification reaction. The protocol covers all steps from the PCR amplification and capture of single-stranded template, its preparation, and the Pyrosequencing assay itself. Once the correct primer stoichiometry has been determined, the assay takes around 2 h for PCR amplification, followed by 15-20 min (per plate) to obtain the genotypes.     

Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction

Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.     

The conversion of wheat RFLP probes into STS markers via the single-stranded conformation polymorphism technique.

We describe a flexible and general strategy for converting a wheat RFLP-based assay into a PCR-based sequence-tagged site (STS), and have applied it to derive markers for a powdery mildew resistance gene present in a wheat-rye translocation. The concept is based on deriving PCR primers that amplify all of the homoeoloci defined by a single-copy cDNA sequence, and separating the resulting mixture of homoeoamplicons via single-stranded conformation polymorphism (SSCP) gels, which are able to detect minor differences between related DNA sequences. After their separation, the individual homoeoamplicons were sequenced and these were used to define nucleotide polymorphisms that could be exploited to design locus-specific PCR primers. In one case, we were able to demonstrate that the assay was allele specific.     

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program—which detects and avoids common SNPs in gene-specific PCR primers—we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.     

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.