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1.  Second-line systemic treatment for advanced cholangiocarcinoma 
Background
Gemcitabine plus platinum (GEM-P) combination chemotherapy is standard treatment for first-line advanced cholangiocarcinoma (aCC). GEM-P first-line therapy reports a progression-free survival (PFS) of 8 months and overall survival (OS) of 11.7 months. Treatment in the second-line setting is less clear. Five-year survival for aCC remains dismal at 5-10%. The purpose of this study was to describe the outcomes with second-line systemic treatment at our institution.
Methods
This study was a single institution retrospective chart review of aCC patients who initiated second-line systemic treatment during 1/1/2009 to 12/31/2012. The primary objective was to evaluate PFS with second-line systemic treatment. Secondary objectives were OS and disease control rate. Second-line systemic regimens were classified into four treatment groups: GEM-P, gemcitabine + fluoropyrimidine (GEM-FU), other FU combination (FU-combo), and others.
Results
Fifty-six patients were included and the majority had intrahepatic aCC. A total of 80% received first-line gemcitabine-based therapy. Second-line therapy consisted of GEM-P (19.6%), GEM-FU (28.6%), FU-combo (37.5%), and others (14.3%). Median PFS was 2.7-month (95% CI, 2.3-3.8 months) with a median OS of 13.8 months (95% CI, 12-19.3 months) and a disease control rate of 50%. No significant difference in survival was identified between the four treatment groups.
Conclusions
This study revealed a 2.7-month PFS, 50% disease control rate, and potential survival benefit with second-line treatment. Options for second-line systemic therapy include GEM-FU, FU-combo, GEM-P if not given in the first-line setting. Targeted therapy with erlotinib or bevacizumab could be considered in addition to chemotherapy.
doi:10.3978/j.issn.2078-6891.2014.072
PMCID: PMC4226829  PMID: 25436118
Cholangiocarcinoma; metastatic; chemotherapy; biotherapy; second-line
2.  Delayed Presentation of DPD Deficiency in Colorectal Cancer 
Case Study 
Mr. D., a 55-year-old male, presented to the medical oncology service with a diagnosis of stage III adenocarcinoma of the sigmoid colon. He presented 7 weeks post sigmoid colectomy with lymph node resection and was initiated on adjuvant chemotherapy with CAPOX (capecitabine [Xeloda] and oxaliplatin [Eloxatin]). Standard dosing was used: oxaliplatin at 130 mg/m2 on day 1 and capecitabine at approximately 2,000 mg/m2/day (rounded to the nearest 500-mg tablet size) for 14 days on and 7 days off (1 cycle = 21 days). A capped body surface area of 2.4 m2 was used, due to the patient’s body habitus.
Adverse Effects 
Mr. D. did not report any complications of therapy during cycle 1, days 1–7, other than grade 1 diarrhea, which was amenable to diphenoxylate/atropine when taken. The next week, he reported significant malaise and fatigue associated with persistent diarrhea occurring every 30 minutes for 5 days. Mr. D. was instructed to go to the emergency room for an immediate evaluation, but he refused.
Mr. D. presented to the clinic in poor condition on day 14 of cycle 1. His diarrhea had increased to grade 3 and was not controlled with either loperamide or diphenoxylate/atropine, though he was not taking his medications as directed. He had been instructed to take two 2-mg loperamide tablets after the first loose stool, followed by 1 tablet of diphenoxylate/atropine 2 hours later. He could then alternate this with loperamide every 2 hours as needed, not to exceed 8 tablets of loperamide per day. Instead, he had taken 2 tablets of loperamide after the first loose stool, but either waited 6 hours to take 1 tablet of diphenoxylate/atropine or otherwise chose not to alternate the medications at all despite continued diarrhea, depending on the day.
Mr. D.’s timing in taking his supportive medications was inconsistent, and his explanations of this timing were not exact. He also reported persistent grade 3 nausea with vomiting for 5 days, which did not improve with ondansetron and prochlorperazine, though he again did not take these consistently. He was advised to alternate ondansetron and prochlorperazine every 4 hours as needed, but only took one or the other medication approximately 3 times per day.
According to Mr. D., his adverse effects initially began on day 9 of cycle 1. He had lost approximately 14 kg (31 lb) during cycle 1. Clinically, he was found to have grade 2 mucositis and grade 1 hand-foot syndrome. At the time of this visit, his absolute neutrophil count was 3,000/ìL, his hemoglobin was 14.4 g/dL, his hematocrit 42.2%, and his platelet count was 139,000/ìL. His kidney function was within the normal range.
Mr. D. refused hospitalization despite the primary team’s recommendation. He also refused to undergo stool sampling for Clostridium difficile. He was given IV fluids along with adjustments in supportive medications, including a prescription for 10% tincture of opium. He was instructed to use 0.6 mL every 6 hours in addition to alternating loperamide with diphenoxylate/atropine as noted previously. He was advised to rinse his mouth with a baking soda solution for relief of his grade 1 mucositis, and alternation of antiemetics every 4 hours was reiterated. He was to return prior to initiation of cycle 2 for further evaluation.
Worsening Symptoms 
The next day, Mr. D.’s wife called the clinic to report that her husband’s diarrhea continued despite the use of tincture of opium and that it was associated with hematochezia. He was also experiencing a worsening of his mucositis, with an associated swelling of the tongue. He was instructed to present to the emergency center, which he did on day 16 of cycle 1. By then, he was found to be febrile at 39.5°C. He was tachycardic, with a heart rate of 126, and he was experiencing significant abdominal pain associated with the diarrhea. The mucositis was worsening, with new odynophagia.
At this time, Mr. D.’s absolute neutrophil count had dropped dramatically to 160/ìL, his hemoglobin was 13.1 g/dL, his hematocrit was 39.2%, and his platelet count was 68,000/ìL. He was admitted to the inpatient service and started on empiric antibiotics. His blood cultures remained negative during hospitalization, but stool cultures were positive for C. difficile. His antimicrobial regimen was deescalated to oral vancomycin once his stool volume decreased. He was treated with an institutional compounded mouthwash of diphenhydramine, aluminum/magnesium hydroxide, and viscous lidocaine for the mucositis, which also slowly improved. He was given a dose of growth factor. Neutropenia eventually resolved, with an absolute neutrophil count of 4,820/ìL on the day of discharge. He was discharged 26 days after initiating cycle 1, at which time his myelosuppression and mucositis were also resolved. Throughout his course, he did not report any neurotoxicity.
DPD Testing 
Due to his severe symptoms of neutropenia, mucositis, and diarrhea, Mr. D. was tested for dihydropyrimidine dehydrogenase (DPD) deficiency. Testing confirmed a heterozygous IVS14+IG>A mutation. For this reason, all further adjuvant therapy was withheld, and he was followed on clinical surveillance only.
PMCID: PMC4114495  PMID: 25089219
3.  The zebrafish reference genome sequence and its relationship to the human genome 
Howe, Kerstin | Clark, Matthew D. | Torroja, Carlos F. | Torrance, James | Berthelot, Camille | Muffato, Matthieu | Collins, John E. | Humphray, Sean | McLaren, Karen | Matthews, Lucy | McLaren, Stuart | Sealy, Ian | Caccamo, Mario | Churcher, Carol | Scott, Carol | Barrett, Jeffrey C. | Koch, Romke | Rauch, Gerd-Jörg | White, Simon | Chow, William | Kilian, Britt | Quintais, Leonor T. | Guerra-Assunção, José A. | Zhou, Yi | Gu, Yong | Yen, Jennifer | Vogel, Jan-Hinnerk | Eyre, Tina | Redmond, Seth | Banerjee, Ruby | Chi, Jianxiang | Fu, Beiyuan | Langley, Elizabeth | Maguire, Sean F. | Laird, Gavin K. | Lloyd, David | Kenyon, Emma | Donaldson, Sarah | Sehra, Harminder | Almeida-King, Jeff | Loveland, Jane | Trevanion, Stephen | Jones, Matt | Quail, Mike | Willey, Dave | Hunt, Adrienne | Burton, John | Sims, Sarah | McLay, Kirsten | Plumb, Bob | Davis, Joy | Clee, Chris | Oliver, Karen | Clark, Richard | Riddle, Clare | Eliott, David | Threadgold, Glen | Harden, Glenn | Ware, Darren | Mortimer, Beverly | Kerry, Giselle | Heath, Paul | Phillimore, Benjamin | Tracey, Alan | Corby, Nicole | Dunn, Matthew | Johnson, Christopher | Wood, Jonathan | Clark, Susan | Pelan, Sarah | Griffiths, Guy | Smith, Michelle | Glithero, Rebecca | Howden, Philip | Barker, Nicholas | Stevens, Christopher | Harley, Joanna | Holt, Karen | Panagiotidis, Georgios | Lovell, Jamieson | Beasley, Helen | Henderson, Carl | Gordon, Daria | Auger, Katherine | Wright, Deborah | Collins, Joanna | Raisen, Claire | Dyer, Lauren | Leung, Kenric | Robertson, Lauren | Ambridge, Kirsty | Leongamornlert, Daniel | McGuire, Sarah | Gilderthorp, Ruth | Griffiths, Coline | Manthravadi, Deepa | Nichol, Sarah | Barker, Gary | Whitehead, Siobhan | Kay, Michael | Brown, Jacqueline | Murnane, Clare | Gray, Emma | Humphries, Matthew | Sycamore, Neil | Barker, Darren | Saunders, David | Wallis, Justene | Babbage, Anne | Hammond, Sian | Mashreghi-Mohammadi, Maryam | Barr, Lucy | Martin, Sancha | Wray, Paul | Ellington, Andrew | Matthews, Nicholas | Ellwood, Matthew | Woodmansey, Rebecca | Clark, Graham | Cooper, James | Tromans, Anthony | Grafham, Darren | Skuce, Carl | Pandian, Richard | Andrews, Robert | Harrison, Elliot | Kimberley, Andrew | Garnett, Jane | Fosker, Nigel | Hall, Rebekah | Garner, Patrick | Kelly, Daniel | Bird, Christine | Palmer, Sophie | Gehring, Ines | Berger, Andrea | Dooley, Christopher M. | Ersan-Ürün, Zübeyde | Eser, Cigdem | Geiger, Horst | Geisler, Maria | Karotki, Lena | Kirn, Anette | Konantz, Judith | Konantz, Martina | Oberländer, Martina | Rudolph-Geiger, Silke | Teucke, Mathias | Osoegawa, Kazutoyo | Zhu, Baoli | Rapp, Amanda | Widaa, Sara | Langford, Cordelia | Yang, Fengtang | Carter, Nigel P. | Harrow, Jennifer | Ning, Zemin | Herrero, Javier | Searle, Steve M. J. | Enright, Anton | Geisler, Robert | Plasterk, Ronald H. A. | Lee, Charles | Westerfield, Monte | de Jong, Pieter J. | Zon, Leonard I. | Postlethwait, John H. | Nüsslein-Volhard, Christiane | Hubbard, Tim J. P. | Crollius, Hugues Roest | Rogers, Jane | Stemple, Derek L.
Nature  2013;496(7446):498-503.
Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
doi:10.1038/nature12111
PMCID: PMC3703927  PMID: 23594743
4.  Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome 
Science (New York, N.Y.)  2010;330(6010):1549-1551.
Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause significant crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison to genomes of related, hemi-biotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding: 1) RXLR effectors and other secreted pathogenicity proteins; 2) enzymes for assimilation of inorganic nitrogen and sulphur; 3) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution towards obligate biotrophy.
doi:10.1126/science.1195203
PMCID: PMC3971456  PMID: 21148394
6.  A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis 
The EMBO Journal  2013;32(18):2430-2438.
Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo-complex, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di-GMP signalling.
A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis
In the plant pathogen X. campestris, the second messenger cGMP controls bacterial virulence and biofilm formation through direct regulation of XC_0249, a novel diguanylate cyclase that synthesises the signalling molecule cyclic di-GMP.
doi:10.1038/emboj.2013.165
PMCID: PMC3770947  PMID: 23881098
biofilm; cyclic di-GMP; signal transduction; virulence; Xanthomonas campestris
7.  Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry 
Nature  2011;477(7363):203-206.
Supergenes are tight clusters of loci that facilitate the co-segregation of adaptive variation, providing integrated control of complex adaptive phenotypes1. Polymorphic supergenes, in which specific combinations of traits are maintained within a single population, were first described for ‘pin’ and ‘thrum’ floral types in Primula1 and Fagopyrum2, but classic examples are also found in insect mimicry3–5 and snail morphology6. Understanding the evolutionary mechanisms that generate these co-adapted gene sets, as well as the mode of limiting the production of unfit recombinant forms, remains a substantial challenge7–10. Here we show that individual wing-pattern morphs in the polymorphic mimetic butterfly Heliconius numata are associated with different genomic rearrangements at the supergene locus P. These rearrangements tighten the genetic linkage between at least two colour-pattern loci that are known to recombine in closely related species9–11, with complete suppression of recombination being observed in experimental crosses across a 400-kilobase interval containing at least 18 genes. In natural populations, notable patterns of linkage disequilibrium (LD) are observed across the entire P region. The resulting divergent haplotype clades and inversion breakpoints are found in complete association with wing-pattern morphs. Our results indicate that allelic combinations at known wing-patterning loci have become locked together in a polymorphic rearrangement at the Plocus, forming a supergene that acts as a simple switch between complex adaptive phenotypes found in sympatry. These findings highlight how genomic rearrangements can have a central role in the coexistence of adaptive phenotypes involving several genes acting in concert, by locally limiting recombination and gene flow.
doi:10.1038/nature10341
PMCID: PMC3717454  PMID: 21841803
8.  The non-obese diabetic mouse sequence, annotation and variation resource: an aid for investigating type 1 diabetes 
Model organisms are becoming increasingly important for the study of complex diseases such as type 1 diabetes (T1D). The non-obese diabetic (NOD) mouse is an experimental model for T1D having been bred to develop the disease spontaneously in a process that is similar to humans. Genetic analysis of the NOD mouse has identified around 50 disease loci, which have the nomenclature Idd for insulin-dependent diabetes, distributed across at least 11 different chromosomes. In total, 21 Idd regions across 6 chromosomes, that are major contributors to T1D susceptibility or resistance, were selected for finished sequencing and annotation at the Wellcome Trust Sanger Institute. Here we describe the generation of 40.4 mega base-pairs of finished sequence from 289 bacterial artificial chromosomes for the NOD mouse. Manual annotation has identified 738 genes in the diabetes sensitive NOD mouse and 765 genes in homologous regions of the diabetes resistant C57BL/6J reference mouse across 19 candidate Idd regions. This has allowed us to call variation consequences between homologous exonic sequences for all annotated regions in the two mouse strains. We demonstrate the importance of this resource further by illustrating the technical difficulties that regions of inter-strain structural variation between the NOD mouse and the C57BL/6J reference mouse can cause for current next generation sequencing and assembly techniques. Furthermore, we have established that the variation rate in the Idd regions is 2.3 times higher than the mean found for the whole genome assembly for the NOD/ShiLtJ genome, which we suggest reflects the fact that positive selection for functional variation in immune genes is beneficial in regard to host defence. In summary, we provide an important resource, which aids the analysis of potential causative genes involved in T1D susceptibility.
Database URLs: http://www.sanger.ac.uk/resources/mouse/nod/; http://vega-previous.sanger.ac.uk/info/data/mouse_regions.html
doi:10.1093/database/bat032
PMCID: PMC3668384  PMID: 23729657
9.  Analyses of pig genomes provide insight into porcine demography and evolution 
Groenen, Martien A. M. | Archibald, Alan L. | Uenishi, Hirohide | Tuggle, Christopher K. | Takeuchi, Yasuhiro | Rothschild, Max F. | Rogel-Gaillard, Claire | Park, Chankyu | Milan, Denis | Megens, Hendrik-Jan | Li, Shengting | Larkin, Denis M. | Kim, Heebal | Frantz, Laurent A. F. | Caccamo, Mario | Ahn, Hyeonju | Aken, Bronwen L. | Anselmo, Anna | Anthon, Christian | Auvil, Loretta | Badaoui, Bouabid | Beattie, Craig W. | Bendixen, Christian | Berman, Daniel | Blecha, Frank | Blomberg, Jonas | Bolund, Lars | Bosse, Mirte | Botti, Sara | Bujie, Zhan | Bystrom, Megan | Capitanu, Boris | Silva, Denise Carvalho | Chardon, Patrick | Chen, Celine | Cheng, Ryan | Choi, Sang-Haeng | Chow, William | Clark, Richard C. | Clee, Christopher | Crooijmans, Richard P. M. A. | Dawson, Harry D. | Dehais, Patrice | De Sapio, Fioravante | Dibbits, Bert | Drou, Nizar | Du, Zhi-Qiang | Eversole, Kellye | Fadista, João | Fairley, Susan | Faraut, Thomas | Faulkner, Geoffrey J. | Fowler, Katie E. | Fredholm, Merete | Fritz, Eric | Gilbert, James G. R. | Giuffra, Elisabetta | Gorodkin, Jan | Griffin, Darren K. | Harrow, Jennifer L. | Hayward, Alexander | Howe, Kerstin | Hu, Zhi-Liang | Humphray, Sean J. | Hunt, Toby | Hornshøj, Henrik | Jeon, Jin-Tae | Jern, Patric | Jones, Matthew | Jurka, Jerzy | Kanamori, Hiroyuki | Kapetanovic, Ronan | Kim, Jaebum | Kim, Jae-Hwan | Kim, Kyu-Won | Kim, Tae-Hun | Larson, Greger | Lee, Kyooyeol | Lee, Kyung-Tai | Leggett, Richard | Lewin, Harris A. | Li, Yingrui | Liu, Wansheng | Loveland, Jane E. | Lu, Yao | Lunney, Joan K. | Ma, Jian | Madsen, Ole | Mann, Katherine | Matthews, Lucy | McLaren, Stuart | Morozumi, Takeya | Murtaugh, Michael P. | Narayan, Jitendra | Nguyen, Dinh Truong | Ni, Peixiang | Oh, Song-Jung | Onteru, Suneel | Panitz, Frank | Park, Eung-Woo | Park, Hong-Seog | Pascal, Geraldine | Paudel, Yogesh | Perez-Enciso, Miguel | Ramirez-Gonzalez, Ricardo | Reecy, James M. | Zas, Sandra Rodriguez | Rohrer, Gary A. | Rund, Lauretta | Sang, Yongming | Schachtschneider, Kyle | Schraiber, Joshua G. | Schwartz, John | Scobie, Linda | Scott, Carol | Searle, Stephen | Servin, Bertrand | Southey, Bruce R. | Sperber, Goran | Stadler, Peter | Sweedler, Jonathan V. | Tafer, Hakim | Thomsen, Bo | Wali, Rashmi | Wang, Jian | Wang, Jun | White, Simon | Xu, Xun | Yerle, Martine | Zhang, Guojie | Zhang, Jianguo | Zhang, Jie | Zhao, Shuhong | Rogers, Jane | Churcher, Carol | Schook, Lawrence B.
Nature  2012;491(7424):393-398.
For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ~1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
doi:10.1038/nature11622
PMCID: PMC3566564  PMID: 23151582
10.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry 
Bentley, David R. | Balasubramanian, Shankar | Swerdlow, Harold P. | Smith, Geoffrey P. | Milton, John | Brown, Clive G. | Hall, Kevin P. | Evers, Dirk J. | Barnes, Colin L. | Bignell, Helen R. | Boutell, Jonathan M. | Bryant, Jason | Carter, Richard J. | Cheetham, R. Keira | Cox, Anthony J. | Ellis, Darren J. | Flatbush, Michael R. | Gormley, Niall A. | Humphray, Sean J. | Irving, Leslie J. | Karbelashvili, Mirian S. | Kirk, Scott M. | Li, Heng | Liu, Xiaohai | Maisinger, Klaus S. | Murray, Lisa J. | Obradovic, Bojan | Ost, Tobias | Parkinson, Michael L. | Pratt, Mark R. | Rasolonjatovo, Isabelle M. J. | Reed, Mark T. | Rigatti, Roberto | Rodighiero, Chiara | Ross, Mark T. | Sabot, Andrea | Sankar, Subramanian V. | Scally, Aylwyn | Schroth, Gary P. | Smith, Mark E. | Smith, Vincent P. | Spiridou, Anastassia | Torrance, Peta E. | Tzonev, Svilen S. | Vermaas, Eric H. | Walter, Klaudia | Wu, Xiaolin | Zhang, Lu | Alam, Mohammed D. | Anastasi, Carole | Aniebo, Ify C. | Bailey, David M. D. | Bancarz, Iain R. | Banerjee, Saibal | Barbour, Selena G. | Baybayan, Primo A. | Benoit, Vincent A. | Benson, Kevin F. | Bevis, Claire | Black, Phillip J. | Boodhun, Asha | Brennan, Joe S. | Bridgham, John A. | Brown, Rob C. | Brown, Andrew A. | Buermann, Dale H. | Bundu, Abass A. | Burrows, James C. | Carter, Nigel P. | Castillo, Nestor | Catenazzi, Maria Chiara E. | Chang, Simon | Cooley, R. Neil | Crake, Natasha R. | Dada, Olubunmi O. | Diakoumakos, Konstantinos D. | Dominguez-Fernandez, Belen | Earnshaw, David J. | Egbujor, Ugonna C. | Elmore, David W. | Etchin, Sergey S. | Ewan, Mark R. | Fedurco, Milan | Fraser, Louise J. | Fajardo, Karin V. Fuentes | Furey, W. Scott | George, David | Gietzen, Kimberley J. | Goddard, Colin P. | Golda, George S. | Granieri, Philip A. | Green, David E. | Gustafson, David L. | Hansen, Nancy F. | Harnish, Kevin | Haudenschild, Christian D. | Heyer, Narinder I. | Hims, Matthew M. | Ho, Johnny T. | Horgan, Adrian M. | Hoschler, Katya | Hurwitz, Steve | Ivanov, Denis V. | Johnson, Maria Q. | James, Terena | Jones, T. A. Huw | Kang, Gyoung-Dong | Kerelska, Tzvetana H. | Kersey, Alan D. | Khrebtukova, Irina | Kindwall, Alex P. | Kingsbury, Zoya | Kokko-Gonzales, Paula I. | Kumar, Anil | Laurent, Marc A. | Lawley, Cynthia T. | Lee, Sarah E. | Lee, Xavier | Liao, Arnold K. | Loch, Jennifer A. | Lok, Mitch | Luo, Shujun | Mammen, Radhika M. | Martin, John W. | McCauley, Patrick G. | McNitt, Paul | Mehta, Parul | Moon, Keith W. | Mullens, Joe W. | Newington, Taksina | Ning, Zemin | Ng, Bee Ling | Novo, Sonia M. | O'Neill, Michael J. | Osborne, Mark A. | Osnowski, Andrew | Ostadan, Omead | Paraschos, Lambros L. | Pickering, Lea | Pike, Andrew C. | Pike, Alger C. | Pinkard, D. Chris | Pliskin, Daniel P. | Podhasky, Joe | Quijano, Victor J. | Raczy, Come | Rae, Vicki H. | Rawlings, Stephen R. | Rodriguez, Ana Chiva | Roe, Phyllida M. | Rogers, John | Rogert Bacigalupo, Maria C. | Romanov, Nikolai | Romieu, Anthony | Roth, Rithy K. | Rourke, Natalie J. | Ruediger, Silke T. | Rusman, Eli | Sanches-Kuiper, Raquel M. | Schenker, Martin R. | Seoane, Josefina M. | Shaw, Richard J. | Shiver, Mitch K. | Short, Steven W. | Sizto, Ning L. | Sluis, Johannes P. | Smith, Melanie A. | Sohna, Jean Ernest Sohna | Spence, Eric J. | Stevens, Kim | Sutton, Neil | Szajkowski, Lukasz | Tregidgo, Carolyn L. | Turcatti, Gerardo | vandeVondele, Stephanie | Verhovsky, Yuli | Virk, Selene M. | Wakelin, Suzanne | Walcott, Gregory C. | Wang, Jingwen | Worsley, Graham J. | Yan, Juying | Yau, Ling | Zuerlein, Mike | Rogers, Jane | Mullikin, James C. | Hurles, Matthew E. | McCooke, Nick J. | West, John S. | Oaks, Frank L. | Lundberg, Peter L. | Klenerman, David | Durbin, Richard | Smith, Anthony J.
Nature  2008;456(7218):53-59.
doi:10.1038/nature07517
PMCID: PMC2581791  PMID: 18987734
11.  Insights into hominid evolution from the gorilla genome sequence 
Nature  2012;483(7388):169-175.
Summary
Gorillas are humans’ closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago (Mya). In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution.
doi:10.1038/nature10842
PMCID: PMC3303130  PMID: 22398555
12.  The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses 
Young, Nevin D. | Debellé, Frédéric | Oldroyd, Giles E. D. | Geurts, Rene | Cannon, Steven B. | Udvardi, Michael K. | Benedito, Vagner A. | Mayer, Klaus F. X. | Gouzy, Jérôme | Schoof, Heiko | Van de Peer, Yves | Proost, Sebastian | Cook, Douglas R. | Meyers, Blake C. | Spannagl, Manuel | Cheung, Foo | De Mita, Stéphane | Krishnakumar, Vivek | Gundlach, Heidrun | Zhou, Shiguo | Mudge, Joann | Bharti, Arvind K. | Murray, Jeremy D. | Naoumkina, Marina A. | Rosen, Benjamin | Silverstein, Kevin A. T. | Tang, Haibao | Rombauts, Stephane | Zhao, Patrick X. | Zhou, Peng | Barbe, Valérie | Bardou, Philippe | Bechner, Michael | Bellec, Arnaud | Berger, Anne | Bergès, Hélène | Bidwell, Shelby | Bisseling, Ton | Choisne, Nathalie | Couloux, Arnaud | Denny, Roxanne | Deshpande, Shweta | Dai, Xinbin | Doyle, Jeff | Dudez, Anne-Marie | Farmer, Andrew D. | Fouteau, Stéphanie | Franken, Carolien | Gibelin, Chrystel | Gish, John | Goldstein, Steven | González, Alvaro J. | Green, Pamela J. | Hallab, Asis | Hartog, Marijke | Hua, Axin | Humphray, Sean | Jeong, Dong-Hoon | Jing, Yi | Jöcker, Anika | Kenton, Steve M. | Kim, Dong-Jin | Klee, Kathrin | Lai, Hongshing | Lang, Chunting | Lin, Shaoping | Macmil, Simone L | Magdelenat, Ghislaine | Matthews, Lucy | McCorrison, Jamison | Monaghan, Erin L. | Mun, Jeong-Hwan | Najar, Fares Z. | Nicholson, Christine | Noirot, Céline | O’Bleness, Majesta | Paule, Charles R. | Poulain, Julie | Prion, Florent | Qin, Baifang | Qu, Chunmei | Retzel, Ernest F. | Riddle, Claire | Sallet, Erika | Samain, Sylvie | Samson, Nicolas | Sanders, Iryna | Saurat, Olivier | Scarpelli, Claude | Schiex, Thomas | Segurens, Béatrice | Severin, Andrew J. | Sherrier, D. Janine | Shi, Ruihua | Sims, Sarah | Singer, Susan R. | Sinharoy, Senjuti | Sterck, Lieven | Viollet, Agnès | Wang, Bing-Bing | Wang, Keqin | Wang, Mingyi | Wang, Xiaohong | Warfsmann, Jens | Weissenbach, Jean | White, Doug D. | White, Jim D. | Wiley, Graham B. | Wincker, Patrick | Xing, Yanbo | Yang, Limei | Yao, Ziyun | Ying, Fu | Zhai, Jixian | Zhou, Liping | Zuber, Antoine | Dénarié, Jean | Dixon, Richard A. | May, Gregory D. | Schwartz, David C. | Rogers, Jane | Quétier, Francis | Town, Christopher D. | Roe, Bruce A.
Nature  2011;480(7378):520-524.
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2. Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.
doi:10.1038/nature10625
PMCID: PMC3272368  PMID: 22089132
13.  Use of Genome Sequence Information for Meat Quality Trait QTL Mining for Causal Genes and Mutations on Pig Chromosome 17 
The newly available pig genome sequence has provided new information to fine map quantitative trait loci (QTL) in order to eventually identify causal variants. With targeted genomic sequencing efforts, we were able to obtain high quality BAC sequences that cover a region on pig chromosome 17 where a number of meat quality QTL have been previously discovered. Sequences from 70 BAC clones were assembled to form an 8-Mbp contig. Subsequently, we successfully mapped five previously identified QTL, three for meat color and two for lactate related traits, to the contig. With an additional 25 genetic markers that were identified by sequence comparison, we were able to carry out further linkage disequilibrium analysis to narrow down the genomic locations of these QTL, which allowed identification of the chromosomal regions that likely contain the causative variants. This research has provided one practical approach to combine genetic and molecular information for QTL mining.
doi:10.3389/fgene.2011.00043
PMCID: PMC3268380  PMID: 22303339
meat quality QTL; pig chromosome 17; integrated analysis
14.  Clustered Coding Variants in the Glutamate Receptor Complexes of Individuals with Schizophrenia and Bipolar Disorder 
PLoS ONE  2011;6(4):e19011.
Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic ‘hub’ genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.
doi:10.1371/journal.pone.0019011
PMCID: PMC3084736  PMID: 21559497
15.  DNA methylation profiling of human chromosomes 6, 20 and 22 
Nature genetics  2006;38(12):1378-1385.
DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.
doi:10.1038/ng1909
PMCID: PMC3082778  PMID: 17072317
17.  Genome-wide and fine-resolution association analysis of malaria in West Africa 
Jallow, Muminatou | Teo, Yik Ying | Small, Kerrin S | Rockett, Kirk A | Deloukas, Panos | Clark, Taane G | Kivinen, Katja | Bojang, Kalifa A | Conway, David J | Pinder, Margaret | Sirugo, Giorgio | Sisay-Joof, Fatou | Usen, Stanley | Auburn, Sarah | Bumpstead, Suzannah J | Campino, Susana | Coffey, Alison | Dunham, Andrew | Fry, Andrew E | Green, Angela | Gwilliam, Rhian | Hunt, Sarah E | Inouye, Michael | Jeffreys, Anna E | Mendy, Alieu | Palotie, Aarno | Potter, Simon | Ragoussis, Jiannis | Rogers, Jane | Rowlands, Kate | Somaskantharajah, Elilan | Whittaker, Pamela | Widden, Claire | Donnelly, Peter | Howie, Bryan | Marchini, Jonathan | Morris, Andrew | SanJoaquin, Miguel | Achidi, Eric Akum | Agbenyega, Tsiri | Allen, Angela | Amodu, Olukemi | Corran, Patrick | Djimde, Abdoulaye | Dolo, Amagana | Doumbo, Ogobara K | Drakeley, Chris | Dunstan, Sarah | Evans, Jennifer | Farrar, Jeremy | Fernando, Deepika | Hien, Tran Tinh | Horstmann, Rolf D | Ibrahim, Muntaser | Karunaweera, Nadira | Kokwaro, Gilbert | Koram, Kwadwo A | Lemnge, Martha | Makani, Julie | Marsh, Kevin | Michon, Pascal | Modiano, David | Molyneux, Malcolm E | Mueller, Ivo | Parker, Michael | Peshu, Norbert | Plowe, Christopher V | Puijalon, Odile | Reeder, John | Reyburn, Hugh | Riley, Eleanor M | Sakuntabhai, Anavaj | Singhasivanon, Pratap | Sirima, Sodiomon | Tall, Adama | Taylor, Terrie E | Thera, Mahamadou | Troye-Blomberg, Marita | Williams, Thomas N | Wilson, Michael | Kwiatkowski, Dominic P
Nature genetics  2009;41(6):657-665.
We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10−7 to P = 4 × 10−14, with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.
doi:10.1038/ng.388
PMCID: PMC2889040  PMID: 19465909
18.  Pig genome sequence - analysis and publication strategy 
BMC Genomics  2010;11:438.
Background
The pig genome is being sequenced and characterised under the auspices of the Swine Genome Sequencing Consortium. The sequencing strategy followed a hybrid approach combining hierarchical shotgun sequencing of BAC clones and whole genome shotgun sequencing.
Results
Assemblies of the BAC clone derived genome sequence have been annotated using the Pre-Ensembl and Ensembl automated pipelines and made accessible through the Pre-Ensembl/Ensembl browsers. The current annotated genome assembly (Sscrofa9) was released with Ensembl 56 in September 2009. A revised assembly (Sscrofa10) is under construction and will incorporate whole genome shotgun sequence (WGS) data providing > 30× genome coverage. The WGS sequence, most of which comprise short Illumina/Solexa reads, were generated from DNA from the same single Duroc sow as the source of the BAC library from which clones were preferentially selected for sequencing. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement the data have been released into public sequence repositories (Genbank/EMBL, NCBI/Ensembl trace repositories) in a timely manner and in advance of publication.
Conclusions
In this marker paper, the Swine Genome Sequencing Consortium (SGSC) sets outs its plans for analysis of the pig genome sequence, for the application and publication of the results.
doi:10.1186/1471-2164-11-438
PMCID: PMC3017778  PMID: 20642822
19.  Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes 
Cancer research  2010;70(2):520-531.
The cyclin dependent kinase (CDK) inhibitors p15, p16, p21 and p27 are frequently deleted, silenced or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development demonstrating that these genes can act as tumor suppressors. Here we describe high-throughput murine leukemia virus (MuLV) insertional mutagenesis screens in mice deficient for one or a combination of two CDK inhibitors. We retrieved 9117 retroviral insertions from 476 lymphomas and find hundreds of loci that are mutated significantly more frequently than expected by chance. Many of these are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also find associations between these loci and gender, age of tumor onset and with lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with SNPs associated with chronic lymphocytic leukemia (CLL) reveals significant overlap between these datasets. Together these data highlight the importance of genetic context within large-scale mutation detection studies and demonstrate a novel use for insertional mutagenesis data in prioritization of disease associated genes resulting from genome-wide association studies.
doi:10.1158/0008-5472.CAN-09-2736
PMCID: PMC2875110  PMID: 20068150
CDK inhibitors; insertional mutagenesis; lymphoma; CLL; Down syndrome
20.  Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity 
Nature genetics  2007;39(3):329-337.
Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately two-fold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we demonstrate that engineered haplodeficiency of IL-2 gene expression not only reduces T cell IL-2 production by two-fold but also mimics the autoimmune dysregulatory effects of the naturally-occurring susceptibility alleles of IL-2. Reduced IL-2 production achieved by both genetic mechanisms correlates with fewer and less functional CD4+CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.
doi:10.1038/ng1958
PMCID: PMC2886969  PMID: 17277778
21.  Genome-wide end-sequenced BAC resources for the NOD/MrkTac☆ and NOD/ShiLtJ☆☆ mouse genomes 
Genomics  2010;95(2):105-110.
Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.
doi:10.1016/j.ygeno.2009.10.004
PMCID: PMC2824108  PMID: 19909804
Bacterial artificial chromosome; NOD/MrkTac; NOD/ShiLtJ; Mouse genome; Non-obese diabetic (NOD); Type 1 diabetes; T1D; Insulin-dependent diabetes; IDD
22.  The genome of the blood fluke Schistosoma mansoni 
Nature  2009;460(7253):352-358.
Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. We report here analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and novel families of micro-exon genes that undergo frequent alternate splicing. As the first sequenced flatworm, and a representative of the lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, while the identification of membrane receptors, ion channels and more than 300 proteases, provide new insights into the biology of the life cycle and novel targets. Bioinformatics approaches have identified metabolic chokepoints while a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.
doi:10.1038/nature08160
PMCID: PMC2756445  PMID: 19606141
23.  Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin 
PLoS Genetics  2009;5(12):e1000759.
An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.
Author Summary
Here we show that tiny DNA lesions can be found in huge amounts of DNA sequence data, similar to finding a needle in a haystack. These lesions identify many new candidates for disease genes associated with birth defects, infertility, and growth. Further, our data suggest that we know very little about what mammalian genes do. Sequencing methods are becoming cheaper and faster. Therefore, our strategy, shown here for the first time, will become commonplace.
doi:10.1371/journal.pgen.1000759
PMCID: PMC2782131  PMID: 20011118
24.  Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution 
PLoS Genetics  2009;5(6):e1000538.
Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15–18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.
Author Summary
Mammalian genomes are remarkably stable (with few exceptions). In humans, wrong recombination events occur quite rarely, manifesting themselves in genomic disorders or cancer. On exceptional occasions, the rate of genome evolution has been accelerated by genome-wide reshuffling events giving rise to some highly derivative karyotypes. The genomes of gibbon species (Hylobatidae) are an example of accelerated genome structural evolution; gibbons display a rate of chromosome evolution 10–20 fold higher than the default rate found in mammals (one chromosome change every 4 million years). As we are interested in investigating the possible genetic causes of this phenomenon, we sequenced a considerable number of chromosomal breakpoints in the northern white-cheeked gibbon genome and analyzed the genomic features of these sites. We observe that the gibbon breakpoints are mostly associated with endogenous retrotransposons called Alus, which are normally abundant in the genomes of primates. Furthermore, our analysis revealed that gibbon Alus have a lower content of methylated CpG when compared to the orthologous human Alus. In mammals, CpG methylation is known to be responsible for keeping retrotransposons in a repressed state and protect genome integrity. We therefore suggest that a glitch in the methylation apparatus might have driven the higher genome recombination in gibbons.
doi:10.1371/journal.pgen.1000538
PMCID: PMC2695003  PMID: 19557196
25.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry 
Bentley, David R. | Balasubramanian, Shankar | Swerdlow, Harold P. | Smith, Geoffrey P. | Milton, John | Brown, Clive G. | Hall, Kevin P. | Evers, Dirk J. | Barnes, Colin L. | Bignell, Helen R. | Boutell, Jonathan M. | Bryant, Jason | Carter, Richard J. | Cheetham, R. Keira | Cox, Anthony J. | Ellis, Darren J. | Flatbush, Michael R. | Gormley, Niall A. | Humphray, Sean J. | Irving, Leslie J. | Karbelashvili, Mirian S. | Kirk, Scott M. | Li, Heng | Liu, Xiaohai | Maisinger, Klaus S. | Murray, Lisa J. | Obradovic, Bojan | Ost, Tobias | Parkinson, Michael L. | Pratt, Mark R. | Rasolonjatovo, Isabelle M. J. | Reed, Mark T. | Rigatti, Roberto | Rodighiero, Chiara | Ross, Mark T. | Sabot, Andrea | Sankar, Subramanian V. | Scally, Aylwyn | Schroth, Gary P. | Smith, Mark E. | Smith, Vincent P. | Spiridou, Anastassia | Torrance, Peta E. | Tzonev, Svilen S. | Vermaas, Eric H. | Walter, Klaudia | Wu, Xiaolin | Zhang, Lu | Alam, Mohammed D. | Anastasi, Carole | Aniebo, Ify C. | Bailey, David M. D. | Bancarz, Iain R. | Banerjee, Saibal | Barbour, Selena G. | Baybayan, Primo A. | Benoit, Vincent A. | Benson, Kevin F. | Bevis, Claire | Black, Phillip J. | Boodhun, Asha | Brennan, Joe S. | Bridgham, John A. | Brown, Rob C. | Brown, Andrew A. | Buermann, Dale H. | Bundu, Abass A. | Burrows, James C. | Carter, Nigel P. | Castillo, Nestor | Catenazzi, Maria Chiara E. | Chang, Simon | Cooley, R. Neil | Crake, Natasha R. | Dada, Olubunmi O. | Diakoumakos, Konstantinos D. | Dominguez-Fernandez, Belen | Earnshaw, David J. | Egbujor, Ugonna C. | Elmore, David W. | Etchin, Sergey S. | Ewan, Mark R. | Fedurco, Milan | Fraser, Louise J. | Fuentes Fajardo, Karin V. | Furey, W. Scott | George, David | Gietzen, Kimberley J. | Goddard, Colin P. | Golda, George S. | Granieri, Philip A. | Green, David E. | Gustafson, David L. | Hansen, Nancy F. | Harnish, Kevin | Haudenschild, Christian D. | Heyer, Narinder I. | Hims, Matthew M. | Ho, Johnny T. | Horgan, Adrian M. | Hoschler, Katya | Hurwitz, Steve | Ivanov, Denis V. | Johnson, Maria Q. | James, Terena | Jones, T. A. Huw | Kang, Gyoung-Dong | Kerelska, Tzvetana H. | Kersey, Alan D. | Khrebtukova, Irina | Kindwall, Alex P. | Kingsbury, Zoya | Kokko-Gonzales, Paula I. | Kumar, Anil | Laurent, Marc A. | Lawley, Cynthia T. | Lee, Sarah E. | Lee, Xavier | Liao, Arnold K. | Loch, Jennifer A. | Lok, Mitch | Luo, Shujun | Mammen, Radhika M. | Martin, John W. | McCauley, Patrick G. | McNitt, Paul | Mehta, Parul | Moon, Keith W. | Mullens, Joe W. | Newington, Taksina | Ning, Zemin | Ling Ng, Bee | Novo, Sonia M. | O'Neill, Michael J. | Osborne, Mark A. | Osnowski, Andrew | Ostadan, Omead | Paraschos, Lambros L. | Pickering, Lea | Pike, Andrew C. | Pike, Alger C. | Pinkard, D. Chris | Pliskin, Daniel P. | Podhasky, Joe | Quijano, Victor J. | Raczy, Come | Rae, Vicki H. | Rawlings, Stephen R. | Rodriguez, Ana Chiva | Roe, Phyllida M. | Rogers, John | Rogert Bacigalupo, Maria C. | Romanov, Nikolai | Romieu, Anthony | Roth, Rithy K. | Rourke, Natalie J. | Ruediger, Silke T. | Rusman, Eli | Sanches-Kuiper, Raquel M. | Schenker, Martin R. | Seoane, Josefina M. | Shaw, Richard J. | Shiver, Mitch K. | Short, Steven W. | Sizto, Ning L. | Sluis, Johannes P. | Smith, Melanie A. | Sohna Sohna, Jean Ernest | Spence, Eric J. | Stevens, Kim | Sutton, Neil | Szajkowski, Lukasz | Tregidgo, Carolyn L. | Turcatti, Gerardo | vandeVondele, Stephanie | Verhovsky, Yuli | Virk, Selene M. | Wakelin, Suzanne | Walcott, Gregory C. | Wang, Jingwen | Worsley, Graham J. | Yan, Juying | Yau, Ling | Zuerlein, Mike | Rogers, Jane | Mullikin, James C. | Hurles, Matthew E. | McCooke, Nick J. | West, John S. | Oaks, Frank L. | Lundberg, Peter L. | Klenerman, David | Durbin, Richard | Smith, Anthony J.
Nature  2008;456(7218):53-59.
DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally employed long (400–800 bp) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intra-species genetic variation. We report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterise four million SNPs and four hundred thousand structural variants, many of which are previously unknown. Our approach is effective for accurate, rapid and economical whole genome re-sequencing and many other biomedical applications.
doi:10.1038/nature07517
PMCID: PMC2581791  PMID: 18987734

Results 1-25 (48)