A Streamlined Random Sequencing Strategy for Finding Coding Exons

The random (shotgun) DNA sequencing strategy is used for most large-scale sequencing projects, including the identification of human disease genes after positional cloning. The principle of the method--sequence assembly from overlap--requires the candidate gene region to be partitioned into 15- to 20-kb pieces (usually λ inserts), themselves randomly subcloned into M13 prior to sequencing with a 6- to 8-fold redundancy. Most often, a time-consuming directed strategy must be invoked to close the remaining gaps. Ultimately, computer-based methods are invoked to locate putative coding exons within the finished genomic sequence. Given the small average size of vertebrate exons, I show here that they can be detected from the computer analysis of the individual runs, much before completion of contiguity. However, the successful assessment of coding potential from the raw data depends on a combination of new sequence masking techniques. When the identification of coding exons is the primary goal, the usual random sequencing strategy can thus be greatly optimized. The streamlined approach requires only a 2- to 2.5-fold sequencing redundancy, can dispense with the subcloning in λ and the closure of gaps, and can be fully automated. The feasibility of this strategy is demonstrated using data from the X-linked Kallmann syndrome gene region.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.     

Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis

Sequencing targeted DNA regions in large samples is necessary to discover the full spectrum of rare variants. We report an effective Illumina sequencing strategy utilizing pooled samples with novel quality (Srfim) and filtering (SERVIC ⁴ E) algorithms. We sequenced 24 exons in two cohorts of 480 samples each, identifying 47 coding variants, including 30 present once per cohort. Validation by Sanger sequencing revealed an excellent combination of sensitivity and specificity for variant detection in pooled samples of both cohorts as compared to publicly available algorithms.     

Detection of point mutations associated with genetic diseases by an exon scanning technique

A major challenge in genetics is identifying the basis of human heritable disease. We describe an "exon scanning" technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the beta-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline----glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis.     

外显子组测序在人类疾病中的应用

据估计,约85%的人类遗传变异集中在蛋白编码区,因此对全部的蛋白编码区（外显子组）进行重测序,可以快速、有效地鉴定人类疾病遗传变异。以往鉴定孟德尔遗传病的致病基因多采用连锁分析结合候选定位克隆的方法,不仅耗时长,而且成功率低。2009年,科学家第一次应用外显子组测序在4名弗里曼谢尔登综合征（常染色体显性遗传病）中发现了位于MYH3中的点突变,显示出外显子组测序在孟德尔遗传病致病基因鉴定中的强大功效。就复杂疾病而言,传统的关联研究,包括全基因组关联研究（GWAS）,虽然鉴定了大量的常见变异,但对低频变异和罕见变异的检测能力十分有限;深度测序的发展为解决上述问题提供了良好的契机。本文就外显子组测序在人类疾病中的应用作一简要综述。     

Exoquence DNA sequencing.

We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.     

Octamer-primed cycle sequencing using dye-terminator chemistry.

Octamer Sequencing Technology, OST, is a method of DNA sequencing using single octamer oligonucleotides to prime cycle sequencing reactions. This sequencing strategy is faster than a traditional primer-walking strategy, since access to this optimized octamer library eliminates delays associated with designing and synthesizing gene specific primers. In this report, OST has been optimized for fluorescent, dye-terminator cycle sequencing reactions to facilitate parallel processing of samples. The successful adaptation of OST to an automated sequencing platform and the design of and access to an octamer library are critical steps towards developing an efficient 'closed-loop' DNA sequencing system.     

Sequencing data of the antiglioma antibody MUC 2-63 and strategy for construction of chimeric antibodies

The murine monoclonal antibody MUC 2-63 detects a surface antigen expressed in human gliomas as demonstrated by means of immunohistochemistry and Western blot. In addition, MUC 2-63 has been successfully used in vivo for radioimaging in nude mice bearing glioma xenografts and in glioma patients. The genes coding for the variable regions of MUC 2-63 were isolated for construction of antibody derivatives with reduced immunogenic effects and of reduced size. DNA and protein sequencing data and strategy for construction of chimeric antibodies and single-chain Fv fragments are presented.     

用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

全基因组测序及其在遗传性疾病研究及诊断中的应用

最近,随着测序成本的不断降低,数据分析策略的不断提升,全基因组测序（whole-genome sequencing,WGS）已经在癌症、孟德尔遗传病、复杂疾病的致病基因检测中得到了一定运用,并逐步走向了临床诊断。全基因组测序不但可以检测编码区和非编码区的点突变（SNVs）和插入缺失（InDels）,还可以在全基因组范围内检测拷贝数变异（copy number variation,CNV）以及结构变异（structure variation,SV）。本文详细地介绍了全基因组测序的标准生物信息分析流程与方法,及其在疾病研究、临床诊断中的应用,并对全基因组测序在医学遗传学中的应用与研究进展,以及数据分析方面面临的挑战进行了概述。     

高通量DNA甲基化数据的处理和分析方法

DNA甲基化作为一种表观遗传学修饰,在调控基因表达、X染色体失活、印记基因等方面都发挥着重要的作用.不同的DNA甲基化的预处理方法结合二代测序产生了大量的高通量甲基化数据,这些数据的存储、处理和分析是当前亟需解决的问题.在本文中,总结了目前存在的三种高通量DNA甲基化检测技术（限制性内切酶法,亲和纯化法,重亚硫酸盐转换法）,以及针对这些技术产生的高通量数据开发的存储、处理和分析工具.另外,还注重介绍了单碱基水平的DNA甲基化检测技术,BS-Seq的测序原理、数据处理流程以及后续的分析工具.     

Translational database selection and multiplexed sequence capture for up front filtering of reliable breast cancer biomarker candidates

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.     

Genomic alignment

As sequencing techniques become increasingly efficient, the average length of a sequence is bound to grow. Traditional sequence-comparison algorithms can either compare DNA or protein, but not a mixture, which is actually a common situation. Most obtained DNA sequences contain coding regions, and it is more reliable to compare the coding regions as protein than just as DNA.A heuristic algorithm is presented that can compare DNA with both coding and noncoding regions, but that also can compare multiple reading frames and determine which exons are homologous.A program, GenAl (Genomic Alignment), was developed that implements the algorithm. Its use is demonstrated on two retroviruses. Correspondence to: J. Hein     

Genomic sequencing by ligation-mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.     

一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

Strategy and methods for directly sequencing cosmid clones

The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.     

Sequencing the DNA of recombinant chromosomes

With contemporary molecular cloning and DNA sequencing techniques, deriving the primary structure of higher cell genes is now routine. This publication reviews the chemical DNA sequencing method, and suggests strategies for sequencing recombinant DNA, whether arising naturally by chromosome rearrangement in vivo or created experimentally by gene splicing in vitro. One such strategy prepares end-labeled restriction fragments from an inserted or rearranged DNA region, for sequencing by the chemical method. Another maps the point at which the sequences of two related DNA regions diverge, and indicates which restriction endonucleases would be useful for sequencing across that point. These techniques can facilitate sequencing of DNA integrations, excisions, translocations, inversions, and introns in cloned chromosomal segments.     

固态纳米孔:下一代DNA测序技术——原理、工艺与挑战

固态纳米孔测序技术作为新兴的第四代DNA测序技术,具有低成本、高读长、易集成等优势.如今,随着半导体工艺技术的飞速发展,小型化、高速度、大通量的纳米孔测序芯片的实现成为可能.相比传统的测序技术,固态纳米孔测序技术在成本、速度等方面有着十分巨大的优势.然而,作为一种新兴的测序技术,固态纳米孔在制造、测序、集成等方面也存在着诸多挑战.本文主要介绍了纳米孔测序技术的原理、制备工艺和面临的挑战,并展望了未来纳米孔测序技术的发展前景.     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.