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full solution to next generation sequencing data analysis. Currently, it consists of a new alignment tool (SOAPaligner/soap2) [4], a re-sequencing consensus sequence builder (SOAPsnp) [4], an indel finder ( SOAPindel ), a structural variation scanner ( SOAPsv) and a de novo short reads assembler ( SOAPdenovo ) [7].
Cloud Computing Hardware: Provides a stable and efficient platform for storage, processing, and analysis of massive bioinformatics data, allowing users to perform extensive bioinformatics analyses.
To support the wet lab and dry lab, BGI has professional team from various backgrounds, including life science, computer science and management. More than 4000 employees work in BGI; about 1000 people are in bioinformatics research group. All the staffs are well trained and examined before they could access to the real data.
Disease studies in BGI
For disease study, the primary means for disease gene identification has been through traditional positional cloning strategies. The scope of this method is limited, especially in situations where there are small family sizes, locus heterogeneity, substantially reduced reproductive fitness and an abundance of candidate genes present in the mapped region. However, a recently developed combinational strategy using whole-exome capture and high-throughput sequencing technology provides a powerful and affordable means to identify causative genes even in these difficult cases. Concerning about next generation sequencing services, BGI provides a variety of genomic solutions to meet your research needs. BGI's goal of building a library of digital life produces new collaborations and partnerships worldwide, which create efficient, accurate, and reliable sequencing services to support all types of genomic research.
The sequencing services platform BGI provides:
De novo Whole Genome Sequencing: Initial sequencing that results in the primary genetic sequence of animals and plants.
Whole Genome Resequencing: Resequencing of human, animal, and plant genomes where a reference sequence is available.
RNA Sequencing: Sequence data for the study of all transcriptional activities, from both coding and non-coding genomic regions, in any organism.
Epigenomics: Global analysis of epigenetic changes across the entire genome.
Others: Sequencing Disease Research, Microbial Sequencing, BACs, fosmids, and chloroplast and mitochondrial genomes.
Whole genome Re-sequencing
Case one : 1000 genome project
The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the rela-
tionship between genotype and phenotype. The results present in Nature 2010 of the pilot phase of the project were designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms.
As the main sponsors, Sanger Institute, Broad Institute and BGI undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. The study described the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. The study also showed that over 95% of the currently accessible variants found in any individual were present in this data set. On average, each person was found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. These methods and public data will support the next phase of human genetic research [2].
Case two: Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing large scale validity study. The study is to validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results were available from 753 preg-nancieswith the 8-plex sequencing protocol and from314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100%sensitivity and 97.9%specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative
predictive value of 96.9%. In conclusion, multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided [1].
Exome sequencing
Exome is the ~1% of the human genome (human exome ~30Mb) that is the most functionally relevant and most likely to cause phenotypes. Exome sequencing is an innovative technique, which targets exon region. Compared with whole genome resequencing, exome sequencing allows us to sequence more individuals with a higher per-individual depth within the same budget, thus it is much more cost-effective. Ever since the first application of exome sequencing on the study of Miller syndrome [9], ex-ome sequencing has proven successful at uncovering novel causative mutations in genetic disorders such as Kabuki syndrome, severe brain malformations [8], etc. These findings demonstrate exome sequencing is applicable to find pathogenetic/susceptibility genes of human diseases.
Currently, BGI has successfully completed a lot of projects of exome sequencing, such as sequencing 200 exomes of individuals with Danish ancestry, which uncover more deleterious rare variants than expected [5], sequencing the 50 exomes of Tibetans, which identify a number of genes underlying the adaptation to a high-altitude environment [12] and ongoing application of exome sequencing at Asian gastric cancer projects.
Case one: 1000 Mendelian Disorders Project. Mendelian disorders are diseases caused by mutation of single gene. The mutation can occur either on a single chromosome or both of a pair of homologous chromosomes. So far, about 5000 Mendelian disorders have been identified in humans. Over the past several decades, the primary techniques for identifying genes correlated to Mendelian disorders have been positional cloning, physical mapping, and can-didate-gene sequencing. Recently, with the development of next-generation sequencing technology, ex-ome sequencing has been employed and has proven to be a powerful approach to identify the genetic basis of Mendelian disorders.
1000 Mendelian Disorders project was initiated in 2010 May in BGI, and the first paper got published was in 2010 December in the journal of Brain [11]. In this paper, the research group sequenced the whole exome of four patients in a Chinese four-generation spinocerebellar ataxia family and identified a mis-sense mutation, c.1550T-G transition (L517W), in exon 10 of TGM6. This change is at a highly conserved position, is predicted to have a functional impact, and completely cosegregated with the pheno-
type. The exome results were validated using linkage analysis. The mutation identified using exome sequencing was located in the same region (20p13-12.2) as that identified by linkage analysis, which cross-validated TGM6 as the causative spinocerebellar ataxia gene in this family. The result also showed that the causative gene could be mapped by a combined method of linkage analysis and sequencing of one sample from the family. The further confirmation was achieved by identifying another missense mutation c.980A-G transition (D327G) in exon seven of TGM6 in an additional spinocerebellar ataxia family, which also co-segregated with the phenotype. Both mutations were absent in 500 normal unaffected individuals of matched geo-graphical ancestry. The finding of TGM6 as a novel causative gene of spinocerebellar ataxia illustrates whole-exome sequencing of affected individuals from one family as an effective and cost efficient method for mapping genes of rare Mendelian disorders and the use of linkage analysis and exome sequencing for further improving efficiency.
Case two: Cancer research
BGI has applied the exome capture technology to complex diseases research since 2008 and has completed more than 3000 exome samples. As a major member of International Cancer Genome Consortium (ICGC), BGI has established wide cooperation with research institutes, hospitals and pharmaceutical corporations in all over the world and is calling for collaboration on discovery of prevalent and rare mutations in any complex diseases. BGI has taken over the sequencing project of Chinese gastric cancer samples in order to find the disease related variations and mutations among large population. Currently, BGI has completed exom sequencing and analysis of the data; the results manuscripts have been submitted to top journal and under review. Cancer study design is shown in the table 1.
RNA sequencing and small RNA sequencing. RNA sequencing is used to study all RNA molecules, including mRNAs, rRNAs, tRNAs, and other noncoding RNAs produced in one or a population of cells. With next generation sequencing technology, RNA sequencing can be applied to the total set of transcripts in a given organism, or to the specific subset of transcripts present in a particular cell type. This allows the identification of regulatory RNAs, annotation of coding SNPs, determination of the relative abundance of transcripts, and more.
Small RNA sequencing is able to cover almost every kind of small RNAs, including miRNAs, siR-NAs, piRNAs, rRNAs, tRNAs, snRNAs, snoRNAs, repeat associated small RNAs and degraded tags of exon or intron. Deep sequencing used HiSeq TM 2000 is the most reliable method to identify and profile small RNAs.
Table 1
Research design Deliverables
Exome sequencing 100 tumor and 100 normal tissue; 50X /sample find Indel, Shared mutation (frequency >3%)
Whole genome sequencing 10 groups (blood+ primary tumor tissue + metastasis tissue) 30X per sample find SV, Virus integrations or rearrangements, Shared mutation (frequency >3%)
Cell line sequencing whole genome sequencing 50X 170-800bp PE; 20X 2k-40kbp PE; find point mutations ,SV, novel sequence by assembly
Single-cell sequencing 50X exome of 20 normal and 100 tumor single cells; find point mutations and mutation frequency spectrum
Case one: Integrated profiling of microRNAs and mRNAs - microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma.
The purpose of this study was to systematically profile the expression of both mRNA and miRNA genes in clear cell renal cell carcinoma (ccRCC) using massively parallel sequencing technology. The expression of mRNAs and miRNAs were analyzed in tumor tissues and matched normal adjacent tissues obtained from 10 ccRCC patients without distant me-tastases. In a prevalence screen, some of the most interesting results were validated in a large cohort of ccRCC patients. The study has found a total of 404 miRNAs and 9,799 mRNAs were detected to be differentially expressed in the 10 ccRCC patients. BGI also identified 56 novel miRNA candidates in at least two samples. In addition to confirming that canonical cancer genes and miRNAs play pivotal roles in ccRCC development, promising novel candidates (such as PNCK and miR-122) without previous annotation in ccRCC carcinogenesis were also discovered in this study. Pathways controlling cell fatesand cell communication were found to be significantly more likely to be disrupted in ccRCC. Additionally, the results of the prevalence screen revealed that the expression of a miRNA gene cluster located on Xq27.3 was consistently down-regulated in at least 76.7% of ,50 ccRCC patients. In conclusion, the study provided a two-dimensional map of the mRNA and miRNA expression profiles of ccRCC using deep sequencing technology. The results indicate that the phenotypic status of ccRCC is characterized by a loss of normal renal function, down-regulation of metabolic genes, and upregulation of many signal transduction genes in key pathways. Furthermore, it can be concluded that down-regulation of miRNA genes clustered on Xq27.3 is associated with ccRCC [13].
Case two: Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer. The study used genome-wide serum miRNA expression analysis to investigate the role of serum miRNA in predicting prognosis of non-small-cell lung cancer
(NSCLC). In the discovery stage, Solexa sequencing followed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays was used to test the difference in levels of serum miRNAs between 30 patients with longer survival (alive and mean survival time, 49.54 months) and 30 patients with shorter survival matched by age, sex, and stage (dead and mean survival time, 9.54 months). The detected serum miRNAs then were validated in 243 patients (randomly classified into two subgroups: n=120 for the training set, and n=123 for the testing set). Eleven serum miRNAs were found to be altered more than five-fold by Solexa sequencing between longer-survival and shorter-survival groups, and levels of four miRNAs (ie, miR-486, miR-30d, miR-1 and miR-499) were significantly associated with overall survival. The four-miRNA signature also was consistently an independent predictor of overall survival for both training and testing samples. In conclusion, the four-miRNA signature from the serum may serve as a noninvasive predictor for the overall survival of NSCLC [3].
Metagenomics
Metagenomics is the study of genomes of a whole of all microbes in an environmental sample, which gives information of microbial diversity of environmental samples and direct access to the genomes of numerous uncultivable microorganisms.
To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumi-na-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 Gb of sequence, from faecal samples of 124 European individuals. The
gene set, ~ 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinalmicrobial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and
1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. BGI defines and describes the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively [10].
Perspective
BGI focuses on multi-omics research (Genomics, Transcriptomics, Epigenomics, Proteomics, etc), which has been reVol.utionizing biomedical research and drug discovery. Compared with traditional research methods, multi-omics research allows us to explore the genome, transcriptome, and proteome, more broadly and with greater sensitivity and resolution. BGI has built a rigorous QC system, enabling us to serve as an official service provider and ensuring accurate results at the highest quality standards.
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9. Ng SB, Buckingham KJ, Lee C. et al. Exome sequencing identifies the cause of a mendelian disorder // Nat Genet. - 2010. - Vol. 42. - P. 30-35.
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УДК 615.243:616.12=111
PHARMACOGENOMICS OF ANTICOAGULANT AND ANTIPLATELET AGENTS IN CLINICAL
CARDIOLOGY PRACTICE
© Manolopoulos V.G.
Laboratory of Pharmacology and Clinical Pharmacology, Democritus University of Thrace Medical School,
Dragana Campus, Alexandroupolis, Greece
E-mail: emanolop@med. duth. gr
Coumarin derivatives such as warfarin, acenocoumarol and phenprocoumon constitute the world-wide oral anticoagulant treatment of thromboembolic disorders, while clopidogrel is the most commonly prescribed antiplatelet treatment. Response to therapy to these drugs exhibits significant variation among patients. A great proportion of this variation is due to genetic background of individuals. In the case of coumarin derivatives, variations both in the genes of cytochrome P450 enzyme CYP2C9 and vitamin K reductase VKORC1 influence individual responses to anticoagulant therapy. CYP2C9*2 and *3 variant alleles result in decreased CYP2C9 enzymatic activity affecting coumarin pharmacokinetics, while VKORC1 -1639G>A polymorphism influence pharmacodynamics response to coumarins. It appears that lower doses of coumarins may be best for patients with variations in one or both of these genes and efforts are made to incorporate this knowledge in currently used dosing regiments. Towards this direction, pharmacogenetic-based dosing algorithms are currently tested in large prospective, randomized, pharmacogenetic clinical trials both in Eurpoe and the USA. It is hoped that the results of these trials will provide the solid basis for broadly implementing genotype-guided dosing of anticoagulant therapy in the clinical routine. The antiplatelet agent clopidogrel is a pro-drug which is converted to an active metabolite by the polymorphic enzyme CYP2C19. Several studies in the last 5 years have shown that carriers of variant CYP2C19 alleles (CYP2C19*2 and *3 loss of function alleles) have impaired ability to metabolize clopidogrel to its active metabolite and as a result decreased inhibition of platelet aggregation and increased cardiovascular risk. Additionally, a novel allele named CYP2C19*17 has been associated with increased enzyme transcription and better response to clopidogrel. Carriers of this allele may exhibit improved prevention of thrombotic events, but may also have increased risk of bleeding events. Efforts are made to implement CYP2C19 geno-typing in the clinic prior to clopidogrel prescription. In conclusion, it is expected that the broad implementation of pharmacogenomics in routine cardiology clinical practice can help ameliorate risk of bleeding or thrombotic events and increase effectiveness of both anticoagulant and antiplatelet agents.
Keywords: pharmacogenomics, anticoagulant agents, antiplatelet agents, cardiology.
ФАРМАКОГЕНОМИКА АНТИКОАГУЛЯНТНЫХ И АНТИТРОМБОЦИТАРНЫХ СРЕДСТВ В ПРАКТИКЕ
КЛИНИЧЕСКОЙ КАРДИОЛОГИИ
Манолопулос В.
Лаборатория фармакологии и клинической фармакологии, Университет Демокрита Фракийской медицинской школы, Кампус Драгана, Александрополис, Г реция
Производные кумарина, такие как варфарин, аценокумарол и фенпрокумон являются всемирно известными препаратами антикоагулянтной терапии тромбоэмболических расстройств, а клопидогрель - наиболее часто выписываемый препарат антитромбоцитарной терапии. Чувствительность к терапии этими препаратами существенно отличается у пациентов. Большая часть этих отличий связана с генетическими особенностями метаболизма лекарств. В случае производных кумарина, на индивидуальную чувствительность к антикоагулянтной терапии оказывают влияние различия в генах цитохрома P450 фермента CYP2C9 и витамин-К-редуктазы. Варианты аллеля CYP2C9*2 и *3 отвечают за снижение ферментной активности, влияющей на фармакокинетику кумарина, а полиморфизм VKORC1 -1639G>A влияет на фармакодинамическую чувствительность к кумаринам. Так, для людей с вариациями в одном или обоих этих генах адекватными будут более низкие дозы кумаринов и уже предпринимаются попытки включения этой информации в порядок назначения доз в клиническую практику кардиологов. В этом направлении в настоящее время тестируются посредством крупных проспективных рандоминизиро-ванных фармакогенетических клинических испытаний в Европе и США фармакогенетически обоснованные алгоритмы дозирования препаратов. Предполагается, что результаты этих испытаний обеспечат прочный фундамент для широкого внедрения в клиническую практику индивидуального дозирования, основанного на генотипах метаболизирующих ферментов. Антитромбоцитарный препарат клопидогрель - это вещество, которое превращается в активный метаболит благодаря полиморфному ферменту CYP2C19. Несколько исследований в последние 5 лет показали, что носители варианта аллеля CYP2C19 (потеря функциональных аллелей CYP2C19*2 and *3) обладают сниженной способностью метаболизировать кло-пидогрель до активного метаболита, в результате такие индивиды имеют сниженную ингибицию агрегации тромбоцитов и повышенный риск сердечно-сосудистых осложнений. Аллель CYP2C19*17 также ассоциирован с повышением ферментной транскрипции и большей чувствительностью к клопидогрелю. Носители этого аллеля отличаются лучшей защитой от тромбоэмболий, но в то же время - повышенным риском кровотечения. Предпринимаются усилия по внедрению генотипирова-ния CYP2C19 в клиническую практику при назначении клопидогреля. Широкое внедрение фармакогеномики в кардиологическую клиническую практику может способствовать снижению риска кровотечений или тромбоэмболий и повысить эффективность как антикоагулянтной, так и антитромбоцитарной терапии.
Ключевые слова: фармакогеномика, антикоагулянтные препараты, антитромбоцитарные препараты, кардиология.
