纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

Rapid quantification of DNA libraries for next-generation sequencing

The next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced which assures optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. In addition, we find that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount of library DNA required, increasing the accuracy in quantifying amplifiable DNA, and avoiding amplification bias by reducing or eliminating the need to amplify DNA before sequencing.     

Stepping stones in DNA sequencing

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Methodological improvements of pyrosequencing technology

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.     

Octamer-primed sequencing technology: effects of dNTP supplementation

Octamer-primed sequencing is a directed DNA sequencing strategy that employs the use of a presynthesized octamer primer library. Together with electronic octamer sequencing technology (eOST) it provides a faster, less expensive way to obtain DNA sequence information and can be used as a valuable tool for gap closure in large-scale genomic sequencing. In this paper we discuss the effect of dGTP/TTP supplementation on octamer sequencing. We show that addition of 75 µM dGTP and 5 µM TTP can improve the sequencing success rate by increasing the length and accuracy of generated sequence information. We also discuss the effect of template base composition immediately downstream of the octamer primer on the outcome of octamer sequencing.     

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.     

Statistical analysis of DNA sequencing data (1): accuracy test of DNA data by partial re-sequencing.

To qualify DNA data, we have developed a statistical method of deciding whether the DNA data has an acceptable accuracy in sequencing process. The method is to test the probability of sequencing errors, based on partial re-sequencing. The method was successfully applied to a yeast mitochondrial DNA which is previously sequenced (1). The analysis indicates that the entire sequence is very accurate although we found one base change error on the ND1 gene sequence data by a partial re-sampling. This method is applicable to any DNA data.     

A method for counting PCR template molecules with application to next-generation sequencing

Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.     

Uniform Band Intensities in Fluorescent Dye Terminator Sequencing

Abstract

The use of Cyanine dye (Cy^TM 5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase^TM DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Assignment of position-specific error probability to primary DNA sequence data.

DNA sequence predicted from polyacrylamide gel-based technologies is inaccurate because of variations in the quality of the primary data due to limitations of the technology, and to sequence-specific variations due to nucleotide interactions within the DNA molecule and with the gel. The ability to recognize the probability of error in the primary data will be useful in reconstructing the target sequence of a DNA sequencing project, and in estimating the accuracy of the final sequence. This paper describes the use of linear discriminant analysis to assign position-specific probabilities of incorrect, over- and under-prediction of nucleotides for each predicted nucleotide position in primary sequence data generated by a gel-based DNA sequencing technology. Using this method, most of the error potential in primary sequence data can be assigned to a limited number of discrete positions. The use of probability values in the sequence reconstruction process, and in estimating the accuracy of consensus sequence determination is described.     

Discovery of single nucleotide polymorphisms and mutations by pyrosequencing

Comparative genomics, analyzing variation among individual genomes, is an area of intense investigation. DNA sequencing is usually employed to look for polymorphisms and mutations. Pyrosequencing, a real-time DNA sequencing method, is emerging as a popular platform for comparative genomics. Here we review the use of this technology for mutation scanning, polymorphism discovery and chemical haplotyping. We describe the methodology and accuracy of this technique and discuss how to reduce the cost for large-scale analysis.     

基于全基因组测序的法医单核苷酸多态性系谱推断研究

目的 通过全基因组测序（whole genome sequencing，WGS）获得高密度单核苷酸多态性（single nucleotide polymorphism，SNP）分型数据，评估分型准确性，研究建立WGS数据用于法医SNP系谱推断的方法。方法 通过华大MGISEQ-200RS测序平台对样本进行深度为30×的WGS，从测序数据中提取Wegene GSA芯片中的645 199个常染色体SNP位点，质控过滤后运用IBS/IBD算法计算预测亲缘关系，并对样本的族群来源进行分析。结果 从测序数据中提取的SNP分型与Wegene GSA芯片分型的一致率大于99.62%。测序获得的SNP数据使用IBS算法可预测1~4级亲缘关系，4级亲缘预测置信区间准确性达100%，使用IBD算法可预测1~7级亲缘关系，7级亲缘预测为有亲缘关系的准确性达100%，通过高深度WGS数据获取的SNP系谱推断能力与芯片预测结果无显著差异。同时，WGS数据用于族群推断与调查结果一致。结论 WGS技术可应用于法医SNP系谱推断，为案件侦破提供线索。     

An extended boiling method for small-scale preparation of plasmid DNA tailored to long-range automated sequencing

Automated fluorescence sequencing depends on high-quality plasmid DNA, which is conveniently prepared by minipreparation procedures. While those procedures are effective for high-copy number plasmids, purity and yields of low-copy number plasmids are often not sufficient to achieve reasonable sequencing results. Here, we describe a reproducible and cheap procedure for the small-scale preparation of plasmid DNA, which is based on the original Holmes and Quigley protocol, comprising a boiling and two selective precipitation steps. Besides various other modifications, this procedure utilizes polyethylene glycol (PEG) precipitation as a key step to further purify plasmid DNA tailored to automated fluorescence sequencing. Independent of the plasmid size and copy number, the modified procedure yields plasmid DNA, which gives average reading lengths of 800 and more bases with a standard fluorescence cycle sequencing protocol. To demonstrate the efficiency and reproducibility of the method, sequencing data of various human interleukin-6 gene variants cloned in different vectors are presented. This procedure offers an economical alternative to commercial miniprep kits, utilizing silica resins or anion-exchanger matrices and, moreover, is more reliable and consistent with respect to reading lengths and accuracy in automated fluorescence sequencing.     

A simple method for direct automated sequencing of PCR fragments.

A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry. Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products. The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators. The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method.     

DNA 池结合DHPLC 和直接测序技术在江豚SNPs 检测中的应用

选取江豚基因组中的2 个已知单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点,通过PCR 扩增,将PCR 产物按基因频率不同制备成0 ～ 50% 的11 个DNA 池(DNA pool),用于变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)和直接测序分析,以探讨DNA 池中基因频率的最低要求。结果显示,当稀有等位基因的基因频率不少于5% 时可在DHPLC 检测过程中明显分辨;而利用DNA 池进行直接测序时的基因频率则需达到10% 。这提示,为保证DHPLC 分析的准确性和可靠性,制备DNA 池时等摩尔DNA 混合的个体数最好不超过10 个。DNA 池结合DHPLC 技术的高效性与准确性可在大规模的SNPs 位点筛选中发挥作用。