Single-nucleotide polymorphism analysis by pyrosequencing

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.     

Genotyping of Snps in a polyploid genome by pyrosequencing

Single-nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations, and they have become increasingly popular markers for association studies. Allelic discrimination of the mostly binary SNPs has been reported for diploid species, mainly the human, but not for polyploid genomes such as the agriculturally important crops. In the present study, we analyzed the applicability of pyrosequencing to genotyping SNPs in tetraploid potatoes. Out of 94 polymorphic loci tested, 76 (81%) proved to be amenable to allelic discrimination by pyrosequencing. An additional locus could be genotyped by the addition of an ssDNA binding protein to the pyrosequencing reaction. Of the remaining 17 loci, two failed because of the presence of paralogs in the genome, while in the other cases, self-annealing of the primer or template at the low reaction temperature (28 degrees C) employed in pyrosequencing rendered allelic discrimination impossible. The quantitative precision ofpyrosequencing was found to be similar to that of conventional dideoxy sequencing and single-nucleotide primer extension. Exceptfor some sequencespecific limitations, pyrosequencing appears to be an appropriate method for genotying SNPs in polyploid species because it is possible to distinguish not only between homoand heterozygosity but also between the different heterozygous states.     

焦磷酸测序测定SNP的常见问题与解决方法

目的:探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法:以VKORC1基因1639 GA位点、CYP2C19基因636 GA位点及UGT1A1基因TA重复序列(TA)6(TA)7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果:通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 GA位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列(TA)6(TA)7多态性的分型准确性。结论:本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

Comparison between MassARRAY and pyrosequencing for CYP2C19 and ABCB1 gene variants of clopidogrel efficiency genotyping

Clopidogrel is one of the most frequently used drugs in patients to reduce cardiovascular events. Since patients with different genetic variations respond quite differently to clopidogrel therapy, the related genetic testing plays a vital role in its dosage and genetic testing related to clopidogrel therapy is currently considered as routine test worldwide. In this study, we aim to use two different methods MALDI-TOF mass spectrometry and pyrosequencing to detect gene variant of CYP2C19 and ABCB1. Six single nucleotides polymorphisms (SNP) within CYP2C19 (*2, *3, *4, *5, *17) and ABCB1 C3435T in 458 Chinese Han patients were determined using both MassARRAY and Pyrosequencing. Sanger sequencing was used for verification. Results of both methods were analyzed and compared. Allele frequencies of each SNP and distribution of different genotypes were calculated based on the MassARRAY and Sanger sequencing results. Both methods provided 100% call rates for gene variants, while results of six samples were different with two methods. With Sanger sequencing as the reference results, MassARRAY generated all the same results. The minor allele frequencies of the above six SNPs were 27.1% (CYP2C19*), 5.9% (CYP2C19*3), 0% (CYP2C19*4), 0% (CYP2C19*5), 1.1% (CYP2C19*17), 40.9% (ABCB1), respectively. MassARRAY provides accurate clopidogrel related genotyping with relatively high cost-efficiency, throughput and short time when compared with pyrosequencing.     

三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用

为了避免四酶焦测序体系中由于三磷酸腺苷双磷酸酶(apyrase)造成的测序结果偏差, 文章建立了一种定量性能好的无三磷酸腺苷双磷酸酶的三酶焦测序体系。方法是将生物素修饰的DNA模板、荧光素酶和ATP硫酸化酶固定在磁性微球表面进行焦测序反应, 当加入一种dNTP进行焦测序反应完后, 采用磁性分离技术, 除去焦测序反应产生的ATP和剩余的dNTP, 然后加入另一种dNTP进行测序, 按同样的方法去除影响下一轮测序反应的成分, 实现循环测序。此体系能准确判读待测DNA的碱基序列, 且可定量测定单核苷酸序列多态性(SNP)中两种等位基因型的相对比值。文章成功检测了16例正常人和8例唐氏综合征患者样本中21号染色体上两个杂合率较高位点(rs1042917和 rs4818219)的等位基因型比值, 所得结果能够明确说明待测样本中来自于父方和母方的21号染色体数目是否相等。该法具有良好的定量性能, 适合于SNP等位基因型的定量分析, 可以用于唐氏综合征的快速检测。     

焦磷酸测序测定SNP的常见问题与解决方法

目的：探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法：以VKORC1基因1639 G〉A位点、CYP2C19基因636 G〉A位点及UGT1A1基因TA重复序列（TA）6〉（TA）7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果：通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 G〉A位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列（TA）6〉（TA）7多态性的分型准确性。结论：本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

Background

Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.

Methods

To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.

Results

The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.

Conclusions

Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.     

Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.     

SNPs discovery and CAPS marker conversion in soybean

To discover new SNPs and develop an easy assay method in soybean, we compared the high-throughput pyrosequencing ESTs with whole genome sequences in different soybean varieties and identified 3899 SNPs. Transitions were found to be much more frequent than transversions in these SNPs. We found that SNPs were widely distributed in the soybean genome, targeting numerous genes involved in various physiological and biochemical processes influencing important agronomic traits. A set of 16 SNPs were validated in nine soybean varieties, and seven SNPs were converted into CAPS. From functional gene association analysis, the marker CAPS282 on the 3′-UTR of gene Glyma07g03490 was identified as associated with 100-seed weight in soybean. The SNP discovery and CAPS markers conversion system developed in this study is fast and cost effective, and holds great promise for molecular-assisted breeding of soybean.     

Genome-wide polymorphism and comparative analyses in the white-tailed deer (Odocoileus virginianus): a model for conservation genomics

The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms.     

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1

Background

Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution.

Methodology/Principal Findings

HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions.

Conclusions/Significance

Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.     

Identification and in silico analysis of functional SNPs of the BRCA1 gene

Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. Also, the genetics of human phenotype variation could be understood by knowing the functions of these SNPs. It is still a major challenge to identify the functional SNPs in a disease-related gene. In this work, we have analyzed the genetic variation that can alter the expression and the function of the BRCA1 gene using computational methods. Of the total 477 SNPs, 65 were found to be nonsynonymous (ns) SNPs. Among the 14 SNPs in the untranslated region, 4 were found in the 5' and 10 were found in the 3' untranslated region (UTR). It was found that 16.9% of the nsSNPs were damaging, by both the SIFT and the PolyPhen servers. The UTR Resource tool suggested that 2 of 4 SNPs in the 5' UTR and 3 of 10 SNPs in the 3' UTR might change the protein expression levels. We identified major mutations from proline to serine at positions 1776 and 1812 of the native protein of the BRCA1 gene. From a comparison of the stabilizing residues of the native and mutant proteins, we propose that an nsSNP (rs1800751) could be an important candidate for the breast cancer caused by the BRCA1 gene.     

Development of Single-Nucleotide Polymorphism- Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.     

Development of genomic resources for the tick Ixodes ricinus: isolation and characterization of single nucleotide polymorphisms

Assessing the genetic variability of the tick Ixodes ricinus—an important vector of pathogens in Europe—is an essential step for setting up antitick control methods. Here, we report the first identification of a set of SNPs isolated from the genome of I. ricinus, by applying a reduction in genomic complexity, pyrosequencing and new bioinformatics tools. Almost 1.4 million of reads (average length: 528 nt) were generated with a full Roche 454 GS FLX run on two reduced representation libraries of I. ricinus. A newly developed bioinformatics tool (DiscoSnp), which isolates SNPs without requiring any reference genome, was used to obtain 321 088 putative SNPs. Stringent selection criteria were applied in a bioinformatics pipeline to select 1768 SNPs for the development of specific primers. Among 384 randomly SNPs tested by Fluidigm genotyping technology on 464 individuals ticks, 368 SNPs loci (96%) exhibited the presence of the two expected alleles. Hardy–Weinberg equilibrium tests conducted on six natural populations of ticks have shown that from 26 to 46 of the 384 loci exhibited significant heterozygote deficiency.     

Improved Detection of Rare HIV-1 Variants using 454 Pyrosequencing

454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.     

BDNF、GRIN1基因与双相情感障碍的关联研究

为了探讨GRIN1和BDNF基因的遗传变异在双相情感障碍疾病中的相关作用, 从GRIN1、BDNF基因上各取2个SNP位点, 采用TaqMan法对100例双相情感障碍患者和100例健康人进行了单核苷酸多态性分析, 比较两组基因型频率的差异, 并使用软件SHEsis进行单体型分析。结果发现GRIN1基因上的rs2301363和hcv1840191与双相情感障碍发病的关联有统计学意义(P<0.05), 它们形成的单倍型T/G在两组人群中的分布差异亦有统计学意义(P<0.05)。而BDNF基因上的rs7103411和rs6265与双相情感障碍发病无统计学意义。实验结果表明GRIN1基因是双相情感障碍的易感基因之一。     

Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n = 4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.