基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.     

Next Generation Sequencing in der diagnostischen Praxis

Next generation sequencing (NGS) allows whole exome or whole genome sequencing for a given patient to be performed timely and at reasonable cost. This diagnostic quantum leap not only has various legal, ethical and economical aspects but will naturally also impact upon patient care. Currently, however, the wide-spread introduction of NGS into routine diagnostics is facing many obstacles. In particular, it is to be expected that NGS will identify a large number of rare variants in a given patient that are of (yet) unknown clinical significance. As a first step towards solving this problem, we introduce the concept of a database that will systematically integrate genotypic and phenotypic information from the German health care context. Not only will this resource be of great scientific value, but the database shall also provide human geneticists with the evidence base necessary for the reliable evaluation of their patient-related sequencing data.     

高通量测序技术在农作物全基因组序列测定中的应用概览

近几年飞速发展的高通量测序技术(next generation sequencing,NGS)在生命科学研究的各个领域充分展现了其低成本、高通量和应用面广等优势。在现代农业生物技术领域,利用高通量测序技术,科学家们不仅能更经济而高效对农作物、模式植物或不同栽培品种进行深入的全基因组测序、重测序,也可以对成百上千的栽培品种进行高效而准确的遗传差异分析、分子标记分析、连锁图谱分析、表观遗传学分析、转录组分析,进而改进农作物的育种技术,加快新品种的育种研究。其中,获得农作物的全基因组序列是其他研究和分析的基础。本文通过介绍近年来发表的一些利用高通量测序技术进行的农作物全基因组测定和组装的工作,展示高通量测序技术在现代农业生物技术领域的广泛前景以及其建立起来的研究基础。     

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose

Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.

Methods

Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.

Results

Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).

Conclusions

Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.     

Impact of Next Generation Sequencing Techniques in Food Microbiology

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety.     

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.     

Mutation Discovery in Regions of Segmental Cancer Genome Amplifications with CoNAn-SNV: A Mixture Model for Next Generation Sequencing of Tumors

Next generation sequencing has now enabled a cost-effective enumeration of the full mutational complement of a tumor genome-in particular single nucleotide variants (SNVs). Most current computational and statistical models for analyzing next generation sequencing data, however, do not account for cancer-specific biological properties, including somatic segmental copy number alterations (CNAs)-which require special treatment of the data. Here we present CoNAn-SNV (Copy Number Annotated SNV): a novel algorithm for the inference of single nucleotide variants (SNVs) that overlap copy number alterations. The method is based on modelling the notion that genomic regions of segmental duplication and amplification induce an extended genotype space where a subset of genotypes will exhibit heavily skewed allelic distributions in SNVs (and therefore render them undetectable by methods that assume diploidy). We introduce the concept of modelling allelic counts from sequencing data using a panel of Binomial mixture models where the number of mixtures for a given locus in the genome is informed by a discrete copy number state given as input. We applied CoNAn-SNV to a previously published whole genome shotgun data set obtained from a lobular breast cancer and show that it is able to discover 21 experimentally revalidated somatic non-synonymous mutations in a lobular breast cancer genome that were not detected using copy number insensitive SNV detection algorithms. Importantly, ROC analysis shows that the increased sensitivity of CoNAn-SNV does not result in disproportionate loss of specificity. This was also supported by analysis of a recently published lymphoma genome with a relatively quiescent karyotype, where CoNAn-SNV showed similar results to other callers except in regions of copy number gain where increased sensitivity was conferred. Our results indicate that in genomically unstable tumors, copy number annotation for SNV detection will be critical to fully characterize the mutational landscape of cancer genomes.     

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.     

Detecting Large Germline Rearrangements of BRCA1 by Next Generation Tumor Sequencing

Molecular Biology - A majority of BRCA1/2 (BRCA) pathogenic variants (PVs) are single nucleotide substitutions or small insertions/deletions. Copy number variations (CNVs), also known as large...     

二代及三代测序技术在遗传学诊断中的应用进展

遗传病的防治是公共卫生领域的重大课题,而明确病因是遗传病防治的重要环节。高通量测序技术（又称二代测序技术）具有高通量、低成本、高准确度的优点,为遗传诊断及咨询提供了直接证据,已成为遗传学检测不可或缺的有力工具;第三代测序也凭借其长读长的独特优势在临床应用中占据一席之地。二代及三代测序技术各有特点,互为补充,临床中针对不同的检测需求有多种类型的测序方案可供选择。基于此,对二代及三代测序技术的原理、分类及其在遗传学诊断中的应用进展做一综述,以期为临床测序方案的选择提供思路和指导。     

Unveiling Distribution Patterns of Freshwater Phytoplankton by a Next Generation Sequencing Based Approach

The recognition and discrimination of phytoplankton species is one of the foundations of freshwater biodiversity research and environmental monitoring. This step is frequently a bottleneck in the analytical chain from sampling to data analysis and subsequent environmental status evaluation. Here we present phytoplankton diversity data from 49 lakes including three seasonal surveys assessed by next generation sequencing (NGS) of 16S ribosomal RNA chloroplast and cyanobacterial gene amplicons and also compare part of these datasets with identification based on morphology. Direct comparison of NGS to microscopic data from three time-series showed that NGS was able to capture the seasonality in phytoplankton succession as observed by microscopy. Still, the PCR-based approach was only semi-quantitative, and detailed NGS and microscopy taxa lists had only low taxonomic correspondence. This is probably due to, both, methodological constraints and current discrepancies in taxonomic frameworks. Discrepancies included Euglenophyta and Heterokonta that were scarce in the NGS but frequently detected by microscopy and Cyanobacteria that were in general more abundant and classified with high resolution by NGS. A deep-branching taxonomically unclassified cluster was frequently detected by NGS but could not be linked to any group identified by microscopy. NGS derived phytoplankton composition differed significantly among lakes with different trophic status, showing that our approach can resolve phytoplankton communities at a level relevant for ecosystem management. The high reproducibility and potential for standardization and parallelization makes our NGS approach an excellent candidate for simultaneous monitoring of prokaryotic and eukaryotic phytoplankton in inland waters.     

第二代测序在检测人类基因突变中的应用

很多的人类疾病与基因突变有关,基因突变在疾病的诊断和治疗中起到了至关重要的作用.第二代高通量测序,其特点为通量高、速度快、成本低,给检测基因突变带来了革命性的变化.该技术检测基因突变的流程简单,研究人员运用全基因组从测序,目标基因组测序以及转录组测序能够实现基因突变的全方位、高准确的检测.     

Increased Probability of Co-Occurrence of Two Rare Diseases in Consanguineous Families and Resolution of a Complex Phenotype by Next Generation Sequencing

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation.     

A Pipeline for the Development of Microsatellite Markers using Next Generation Sequencing Data

Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants.Objective: This research presents a workflow for developing microsatellite markers using genome skimming.Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog.Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR.Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.