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Posts Tagged ‘gene sequencing’


Vassar Street and Main Street, in the new world’s Cambridge, Massachusetts, would be a leading candidate.

According to the article published in Wired Magazine in November 2015 “when the Whitehead got too small for genomicist Eric Lander’s ambitions, he launched a flashier and brasher newcomer next door. The Broad Institute’s gargantuan gleaming glass lobby is filled with early gene-sequencing instruments. Its multimedia screens boast that this is one of the world’s largest gene-sequencing and research factories. The Broad’s strategy is different from that of the Whitehead; instead of concentrating a few in an ultra-exclusive bioclub, Broad bridges MIT, Harvard and most of the hospitals in Boston. Its 2,000 members extend outwards, partnering with tens of thousands of others globally. Those working at the Broad are not averse to commerce; its director alone helped to build Foundation Medicine, Verastem, Millennium, Fidelity Biosciences, Courtagen and Aclara among many other leading companies.

The sixth building on this extraordinary corner, Novartis, focuses on private research, and represents a huge migration from Basel in Switzerland towards the MIT campus, becoming Cambridge’s largest employer. Pfizer, Sanofi, Amgen, Biogen-Idec and hundreds of others cluster nearby. ”

Attracting the best and the brightest, one can change not just a city but the world.

 

Source

http://www.wired.co.uk/magazine/archive/2015/11/ideas-bank/vassar-main-cambridge-massachusetts-innovaton

 

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What comes after finishing the Euchromatic Sequence of the Human Genome?

Author and Curator: Larry H Bernstein, MD, FCAP 

 

 

  1. Finishing the euchromatic sequence of the human genome.

    Oct 21 2004 ; 431(7011): 931-45.   http://dx.doi.org/10.1038/nature03001
    International Human Genome Sequencing Consortium.

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. They then worked to complete a genome sequence with high accuracy and completeness. The result of this is reported in Nature (2004), here cited. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes
PMID: 15496913 [PubMed – indexed for MEDLINE]

Comment in Human genome: end of the beginning. [Nature. 2004]

 

  1. Human genome:  End of the beginning

Lincoln D. Stein
Nature 21 Oct 2004; 431, 915-916 |   http://dx.doi.org/10.1038/431915a

Just over three years ago, a first draft of the human genome sequence had been completed. Gaps and errors remained, but the job of fixing those problems is now largely done.

The featured article in this issue of Nature, entitled “Finishing the euchromatic sequence of the human genome”, has been authored by members of the International Human Genome Sequencing Consortium (IHGSC).  It is the latest, but by no means the last, milestone in this historic project.

Early in 2001, the duelling IHGSC (public) and Celera Corporation (private) groups published papers in Nature2 and Science3describing the completion of so-called ‘draft’ sequences. These sequences have revolutionized molecular biology by largely eliminating the need to clone and sequence genes involved in human health and disease.

But the draft sequences were far from perfect. Some 10% of the so-called ‘euchromatin’ — the gene-rich portion of the genome — and some 30% of the genome as a whole (which includes the gene-poor regions of ‘heterochromatin’), were not disclosed. There were hundreds of thousands of gaps, and there were misassembled regions where portions of the genome were flipped or misplaced. As a result, large-scale analyses of the genome, had to contend with numerous uncertainties and artefacts. For example, studies of the dying remnants of genes that have accumulated mutations that render them non-functional, left the possibility that such a ‘pseudogene’ was a sequencing error.

Since the publication of the drafts, the IHGSC sequencing centers have quietly undertaken a laborious ‘finishing’ process, in which each gap in the draft was individually examined and subjected to a battery of steps involving cloning and resequencing stretches of DNA. The sequence announced today has just 341 gaps remaining, and consists of contiguous runs of sequence averaging 38 million base pairs. The authors estimate that the finished sequence covers 99% of the euchromatic portion of the genome and that the overall error rate is less than 1 error per 100,000 base pairs. This substantially exceeds the original goals for the project.

The finishing procedure roughly doubled the total time and cost of the project. Does it contribute anything new to our understanding of the genome? It does indeed, and to prove the point the authors of the current paper1 describe several large-scale analyses of the genome that would have been difficult to perform on the draft sequence. One analysis studied the processes of gene birth and death. The authors find 1,183 human genes that show evidence of having been recently ‘born’ by a process of gene duplication and divergence. They also find 37 genes that seem to have recently ‘died’ by acquiring a mutation that rendered the gene non-functional. The resulting pseudogene then slowly degrades and disappears.

The authors then use the finished sequence to map out segmental duplications — large regions of the genome that have duplicated. They find 5% of the genome involved in segmental duplications, and the duplications are distributed widely across the chromosomes. The nature and extent of such duplications sheds light on the evolution of the human genome, and is needed for studying the many medically relevant disorders that are involved in segmental duplications.

Another paper in this issue, by She et al.4 (page 927), directly compares the outcomes of this second analysis with results obtained on an unfinished version of the human genome (an improved version of the Celera draft). She et al. find that the draft version artefactually ‘simplifies’ the genome by eliminating many duplicated regions. Their results bear on one of the highly publicized differences between the public and private genome projects. The public project used an older strategy in which the genome was first cloned into bacterial artificial chromosomes (BACs); the clones were then mapped, and each clone was sequenced and their sequences assembled individually. Celera championed an untested technique, ‘whole-genome shotgun’ (WGS), in which the entire genome was shattered into bite-size pieces, sequenced, and then assembled by software in one conceptually simple step.

Celera proved that the WGS technique is both technically feasible and provides a dramatic cost-saving over the clone-by-clone approach. The Celera draft has had a significant impact on the public project. Almost all genome-sequencing projects since then have used some form of WGS. The cautionary results contained in the new papers from the IHGSC1 and She et al.4argue for a hybrid strategy in which WGS is supplemented by a modest amount of BAC cloning and mapping. This would protect draft WGS sequences from some of the ‘simplification’ reported by She et al. and provide the clones needed for finishing selected regions of special interest.

What is next for the human genome project?

1)       Develop the definitive catalogue of protein-coding genes – estimated to be between 20,000 and 25,000.

a)       Natural selection ensures that functional regions are more highly conserved than non-functional ones, so a comparative approach highlights candidate protein-coding regions.

b)       The same approach shows promise for finding other functional elements such as gene promoters, which control the timing and level of expression of genes, and micro-RNAs, which have been implicated as regulatory agents of many developmental processes.

2)       Sequencing the remaining 20% of the genome that lies within heterochromatin, the gene-poor, highly   repetitive sequence that is implicated in the processes of chromosome replication and maintenance.

a)       The repetitiveness ofheterochromatin means that it cannot be tackled using current sequencing methods, and new technologies will have to be developed to attack it.

We are only at the end of the beginning: ahead lies another mountain range that we will need to map out and explore as we seek to understand how all the parts revealed by the genome sequence work together to make life.

References

  1. International Human Genome Sequencing Consortium Nature 431, 931−945 (2004). | Article |
  2. International Human Genome Sequencing Consortium Nature 409, 860−921 (2001). | Article | PubMed | ISI | ChemPort |
  3. Venter, J. C. et alScience 291, 1304−1351 (2001). | Article | PubMed | ISI | ChemPort |
  4. She, X. et alNature 431, 927−930 (2004). | Article |

 

  1. Shotgun sequence assembly and recent segmental duplications within the human genome

Xinwei She1, Zhaoshi Jiang1, Royden A. Clark2, Ge Liu2, Ze Cheng1, et al.
Nature 431, 927-930 (21 Oct 2004) |    http://dx.doi.org/10.1038/nature03062;

Complex eukaryotic genomes are now being sequenced at an accelerated pace primarily using whole-genome shotgun (WGS) sequence assembly approaches. WGS assembly was initially criticized because of its perceived inability to resolve repeat structures within genomes. Here, we quantify the effect of WGS sequence assembly on large, highly similar repeats by comparison of the segmental duplication content of two different human genome assemblies. Our analysis shows that large (> 15 kilobases) and highly identical (> 97%) duplications are not adequately resolved by WGS assembly. This leads to significant reduction in genome length and the loss of genes embedded within duplications. Comparable analyses of mouse genome assemblies confirm that strict WGS sequence assembly will oversimplify our understanding of mammalian genome structure and evolution; a hybrid strategy using a targeted clone-by-clone approach to resolve duplications is proposed.

Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington
Department of Genetics, Case Western Reserve University, Cleveland, Ohio
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland
Applied Biosystems,  and
The Center for the Advancement of Genomics, Rockville, Maryland

 

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Author: Ziv Raviv, PhD

Introduction

Cutaneous melanoma is a type of skin cancer that originates in melanocytes, the cells that are producing melanin. While being the least common type of skin cancer, melanoma is the most aggressive one with invasive characteristics and accounts for the majority of death incidences among skin cancers. Melanoma has an annual rate of 160,000 new cases and 48,000 deaths worldwide. Melanoma affects mainly Caucasians exposed to sun high UV irradiation. Among the genetic factors that characterize the disease, BRAF mutation (V600E) is found in most cases of melanoma (80%).  Awareness toward risk factors of melanoma should lead to prevention and early detection*. There are several developmental stages (I-IV) of the disease, starting from local non-invasive melanoma, through invasive and high risk melanoma, up to metastatic melanoma. As with other cancers, the earlier stage melanoma is being detected, the better odds for full recovery are. Treatment is usually involving surgery to remove the local tumor and its margins, and when necessary also to remove the proximal lymph node(s) that drain the tumor. In high stages melanoma, adjuvant therapy is given in the form of chemotherapy (Dacarbazine and Temozolomide) and immunotherapy (IL-2 and IFN). Until recently no useful treatment was available for metastatic melanoma. However, research efforts had led to the development of two new drugs against metastatic melanoma: Vemurafenib (Zelboraf), a B-Raf inhibitor; and Ipilimumab (Yervoy), a monoclonal antibody that blocks the inhibitory signal of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Both drugs are now available for clinical use presenting good results.

Personalized therapy for melanoma

In an attempt to develop personalized therapies for malignant melanoma, a unique strategy has been taken by the group of Prof. Yardena Samuels at the NIH (now situated at the WIS) to identify recurring genetic alterations of metastatic cutaneous melanoma. The researchers approach employed the collections of hundreds of tumors samples taken from metastasized melanoma patients together with matched normal blood tissues samples. The samples are undergoing exome sequencing for the analysis of somatic mutations (namely mutations that evolved during the progress of the disease to the stage of metastatic melanoma, unlike genomic mutations that may have contribute to the formation of the disease). The discrimination of such tumor related somatic mutations is done by comparison to the exome sequencing of the patient’s matched blood cells DNA. In addition, the malignant cells derived from the removed cancer tissue of each patient are extracted to form a cell line and are grown in culture. These cells are easily cultivate in culture with no special media supplements, nor further genetic manipulations such as hTERT are needed, and are extremely aggressive as determined by various cell culture and in vivo tests. The ability to grow these primary tumor-derived cell lines in culture has a great value as a tool for studying and characterizing the biochemical, functional, and clinical aspects of the mutated genes identified.

In one study [1] Samuels and her colleagues performed this sequencing process for mutation analysis for the protein tyrosine kinase (PTK) gene family, as PTKs are frequently mutated in cancer. Using high-throughput gene sequencing to analyze the entire PTK gene family, the researchers have identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. The most frequent mutations were found in ERBB4, a member of the EGFR/ErbB family of receptor tyrosine kinase (RTK), were 19% of melanoma patients had such mutations. Seven missense mutations in the ERBB4 gene were found to induce increased kinase activity and transformation capability. Melanoma derived cell lines that were expressing these mutant ERBB4 forms had reduced cell growth after silencing ERBB4 by RNAi or after treatment with the ERBB inhibitor Lapatinib. Lapatinib is already in use in the clinic for the treatment of HER2 (ErbB2) positive breast cancers patients. Following this study, a clinical trial is now conducted with this drug to evaluate its effect in cutaneous metastatic melanoma patients harboring mutations in ERBB4.

In another study of this group [2], the scientists employed the exome sequencing method to analyze the somatic mutations of 734 G protein coupled receptors (GPCRs) in melanoma. GPCRs are regulating various signaling pathways including those that affect cell growth and play also important role in human diseases. This screen revealed that GRM3 gene that encode the metabotropic glutamate receptor 3 (mGluR3), was frequently mutated and that one of its mutations clustered within one position. Mutant GRM3 was found to selectively regulate the phosphorylation of MEK1 leading to increased anchorage-independent cell growth and cellular migration. Tumor derived melanoma cells expressing mutant GRM3 exhibited reduced cell growth and migration upon knockdown of GRM3 by RNAi or by treatment with the selective MEK inhibitor, Selumetinib (AZD-6244), a drug that is being testing in clinical trials. Altogether, the results of this study point to the increased violent characteristics of melanomas bearing mutational GRM3.

In a third study, melanoma samples were examined for somatic mutations in 19 human genes that encode ADAMTS proteins [3]. Some of the ADAMTS genes have been suggested before to have implication in tumorigenesis. ADAMTS18, which was previously found to be a candidate cancer gene, was found in this study to be highly mutated in melanoma. ADAMTS18 mutations were biologically examined and were found to induce an increased proliferation of melanoma cells, as well as increased cell migration and metastasis. Moreover, melanoma cells expressing these mutated ADAMTS18 had reduced cell migration after RNAi-mediated knockdown of ADAMTS18. Thus, these results suggest that genetic alteration of ADAMTS18 plays a major role in melanoma tumorigenesis. Since ADAMTS genes encode extracellular proteins, their accessibility to systematically delivered drugs makes them excellent therapeutic targets.

Conclusive remarks

The above illustrated research approach intends to discover frequent melanoma-specific mutations by employing high-throughput whole exome and genome sequencing means. For the most highly mutated genes identified, the biochemical, functional, and clinical aspects are being characterized to examine their relevancy to the disease outcomes. This approach therefore introduces new opportunities for clinical intervention for the treatment of cutaneous melanoma. In addition to the discovery of novel highly mutated genes, this approach may also help determine which pathways are altered in melanoma and how these genes and pathways interact. Finding melanoma-associated highly mutated genes could lead to personalized therapeutics specifically targeting these altered genes in individual melanomas. Along with the opportunity to develop new agents to treat melanoma, the approach takes advantage of existing anti-cancer drugs, utilizing them to treat these mutated genes melanoma individuals. In addition to their potential for therapeutics, the discovery of highly mutated genes in melanoma patients may lead to the discovery of new markers that may assist the diagnosis of the disease. The implications of these screenings findings on other types of cancer bearing common pathways similar to melanoma should be examined as well. Finally, this elegant approach should be adopted in research efforts of other cancer types.

* Special review will be published further in the cancer prevention section of Pharmaceutical Intelligence

References

1. Prickett TD, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR, Cronin JC, Cruz P, Rosenberg SA, Samuels Y (2009) Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 41 (10):1127-1132

2. Prickett TD, Wei X, Cardenas-Navia I, Teer JK, Lin JC, Walia V, Gartner J, Jiang J, Cherukuri PF, Molinolo A, Davies MA, Gershenwald JE, Stemke-Hale K, Rosenberg SA, Margulies EH, Samuels Y (2011) Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma. Nat Genet 43 (11):1119-1126

3. Wei X, Prickett TD, Viloria CG, Molinolo A, Lin JC, Cardenas-Navia I, Cruz P, Rosenberg SA, Davies MA, Gershenwald JE, Lopez-Otin C, Samuels Y (2010) Mutational and functional analysis reveals ADAMTS18 metalloproteinase as a novel driver in melanoma. Mol Cancer Res 8 (11):1513-1525

Related articles on melanoma on this open access online scientific journal:

1.  In focus: Melanoma Genetics. Curator: Ritu Saxena, Ph.D.

2.  In focus: Melanoma therapeutics. Author and Curator: Ritu Saxena, Ph.D.

3.  A New Therapy for Melanoma.  Reporter- Larry H Bernstein, M.D.

4. Thymosin alpha1 and melanoma. Author, Editor: Tilda Barliya, Ph.D.

5. Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Reporter and Curator: Dr. Sudipta Saha, Ph.D.

6. How Genome Sequencing is Revolutionizing Clinical Diagnostics. Reporter: Aviva Lev-Ari, PhD, RN.

7. Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. Curator and Reporter: Stephen J. Williams, Ph.D.

 

 

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