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Local concentrations of mutations are well known in human cancers. However, their three-dimensional spatial relationships in the encoded protein have yet to be systematically explored. We developed a computational tool, HotSpot3D, to identify such spatial hotspots (clusters) and to interpret the potential function of variants within them. We applied HotSpot3D to >4,400 TCGA tumors across 19 cancer types, discovering >6,000 intra- and intermolecular clusters, some of which showed tumor and/or tissue specificity. In addition, we identified 369 rare mutations in genes including TP53, PTEN, VHL, EGFR, and FBXW7 and 99 medium-recurrence mutations in genes such as RUNX1, MTOR, CA3, PI3, and PTPN11, all mapping within clusters having potential functional implications. As a proof of concept, we validated our predictions in EGFR using high-throughput phosphorylation data and cell-line-based experimental evaluation. Finally, mutation-drug cluster and network analysis predicted over 800 promising candidates for druggable mutations, raising new possibilities for designing personalized treatments for patients carrying specific mutations.

The price of whole-genome sequencing is now within the budget of the average American consumer. This has resulted in the commercialization of genome sequencing for a variety of applications, including health-related risk assessment. Direct-to-consumer marketing of personal DNA sequence information uncouples the generation of personal health-related data from the physician-patient relationship. Here, I discuss the status of consumer genomics and the current and potential concerns about bypassing physicians in the analysis and interpretation of personal genomic information and subsequent health care decision-making.

Recent progress in next-generation sequencing has greatly facilitated our study of genomic structural variation. Unlike single nucleotide variants and small indels, many structural variants have not been completely characterized at nucleotide resolution. Deriving the complete sequences underlying such breakpoints is crucial for not only accurate discovery, but also for the functional characterization of altered alleles. However, our current ability to determine such breakpoint sequences is limited because of challenges in aligning and assembling short reads. To address this issue, we developed a targeted iterative graph routing assembler, TIGRA, which implements a set of novel data analysis routines to achieve effective breakpoint assembly from next-generation sequencing data. In our assessment using data from the 1000 Genomes Project, TIGRA was able to accurately assemble the majority of deletion and mobile element insertion breakpoints, with a substantively better success rate and accuracy than other algorithms. TIGRA has been applied in the 1000 Genomes Project and other projects and is freely available for academic use.

SummaryWe report the results of whole-genome and transcriptome sequencing of tumor and adjacent normal tissue samples from 17 patients with non-small cell lung carcinoma (NSCLC). We identified 3,726 point mutations and more than 90 indels in the coding sequence, with an average mutation frequency more than 10-fold higher in smokers than in never-smokers. Novel alterations in genes involved in chromatin modification and DNA repair pathways were identified, along with DACH1, CFTR, RELN, ABCB5, and HGF. Deep digital sequencing revealed diverse clonality patterns in both never-smokers and smokers. All validated EFGR and KRAS mutations were present in the founder clones, suggesting possible roles in cancer initiation. Analysis revealed 14 fusions, including ROS1 and ALK, as well as novel metabolic enzymes. Cell-cycle and JAK-STAT pathways are significantly altered in lung cancer, along with perturbations in 54 genes that are potentially targetable with currently available drugs.

Summary To correlate the variable clinical features of estrogen receptor positive (ER+) breast cancer with somatic alterations, we studied pre-treatment tumour biopsies accrued from patients in a study of neoadjuvant aromatase inhibitor (AI) therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to hematopoietic disorders. Mutant MAP3K1 was associated with Luminal A status, low grade histology and low proliferation rates whereas mutant TP53 associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon AI treatment. Pathway analysis demonstrated mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in ER+ breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumor biology but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumor progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumor, a brain metastasis, and a xenograft derived from the primary tumor. The metastasis contained two de novo mutations and a large deletion not present in the primary tumor, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumor mutations, and displayed a mutation enrichment pattern that paralleled the metastasis (16 of 20 genes). Two overlapping large deletions, encompassing CTNNA1, were present in all three tumor samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared to the primary tumor suggest that secondary tumors may arise from a minority of cells within the primary.

Detection and characterization of genomic structural variation are important for understanding the landscape of genetic variation in human populations and in complex diseases such as cancer. Recent studies demonstrate the feasibility of detecting structural variation using next-generation, short-insert, paired-end sequencing reads. However, the utility of these reads is not entirely clear, nor are the analysis methods with which accurate detection can be achieved. The algorithm BreakDancer predicts a wide variety of structural variants including insertion-deletions (indels), inversions and translocations. We examined BreakDancer's performance in simulation, in comparison with other methods and in analyses of a sample from an individual with acute myeloid leukemia and of samples from the 1,000 Genomes trio individuals. BreakDancer sensitively and accurately detected indels ranging from 10 base pairs to 1 megabase pair that are difficult to detect via a single conventional approach.

Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas ( TCGA) pilot project aims to assess the value of large- scale multi- dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas - the most common type of primary adult brain cancer - and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol- 3- OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

Whole-genome sequencing of the soybean ( Glycine max (L.) Merr. ‘Williams 82’) has made it important to integrate its physical and genetic maps. To facilitate this integration of maps, we screened 3290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the ‘Williams 82’ physical map. SSRs were screened against 3 mapping populations. We found the AAT and ACT motifs produced the greatest frequency of length polymorphisms, ranging from 17.2% to 32.3% and from 11.8% to 33.3%, respectively. Other useful motifs include the dinucleotide repeats AG, AT, and AG, with frequency of length polymorphisms ranging from 11.2% to 18.4% (AT), 12.4% to 20.6% (AG), and 11.3% to 16.4% (GT). Repeat lengths less than 16 bp were generally less useful than repeat lengths of 40–60 bp. Two hundred and sixty-five SSRs were genetically mapped in at least one population. Of the 265 mapped SSRs, 60 came from BAC singletons not yet placed into contigs of the physical map. One hundred and ten originated in BACs located in contigs for which no genetic map location was previously known. Ninety-five SSRs came from BACs within contigs for which one or more other BACs had already been mapped. For these fingerprinted contigs (FPC) a high percentage of the mapped markers showed inconsistent map locations. A strategy is introduced by which physical and genetic map inconsistencies can be resolved using the preliminary 4× assembly of the whole genome sequence of soybean.

The rhesus macaque ( Macaca mulatta ) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.

We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity.

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

Nat. Genet. 48, 827–837 (2016); published online 13 June 2016; corrected after print 20 March 2017 In the version of this article initially published, residue R384 was incorrectly highlighted in the protein model depicted in Figure 7e. The correct residue is R394. The error has been corrected in theHTML and PDF versions of the article.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

The Cancer Genome Atlas (TCGA) Research Network has profiled and analyzed large numbers of human tumors to discover molecular aberrations at the DNA, RNA, protein and epigenetic levels. The resulting rich data provide a major opportunity to develop an integrated picture of commonalities, differences and emergent themes across tumor lineages. The Pan-Cancer initiative compares the first 12 tumor types profiled by TCGA. Analysis of the molecular aberrations and their functional roles across tumor types will teach us how to extend therapies effective in one cancer type to others with a similar genomic profile., National Cancer Institute (U.S.), National Human Genome Research Institute (U.S.)

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%–60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.

SummaryMost mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is “captured” as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Context Whole-genome sequencing is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis. Objective To determine whether whole-genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame. Design, Setting, and Patient We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase fluorescence in situ hybridization (FISH). The case patient was enrolled in an institutional review board–approved protocol, with consent specifically tailored to the implications of whole-genome sequencing. The protocol uses a “movable firewall” that maintains patient anonymity within the entire research team but allows the research team to communicate medically relevant information to the treating physician. Main Outcome Measures Clinical relevance of whole-genome sequencing and time to communicate validated results to the treating physician. Results Massively parallel paired-end sequencing allowed identification of a cytogenetically cryptic event: a 77-kilobase segment from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. Reverse transcription polymerase chain reaction sequencing subsequently validated the expression of the fusion transcript. Novel FISH probes identified 2 additional cases of t(15;17)–negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole-genome sequencing and validation were completed in 7 weeks and changed the treatment plan for the patient. Conclusion Whole-genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant time frame.

Motivation: The expansion of cancer genome sequencing continues to stimulate development of analytical tools for inferring relationships between somatic changes and tumor development. Pathway associations are especially consequential, but existing algorithms are demonstrably inadequate. Methods: Here, we propose the PathScan significance test for the scenario where pathway mutations collectively contribute to tumor development. Its design addresses two aspects that established methods neglect. First, we account for variations in gene length and the consequent differences in their mutation probabilities under the standard null hypothesis of random mutation. The associated spike in computational effort is mitigated by accurate convolution-based approximation. Second, we combine individual probabilities into a multiple-sample value using Fisher–Lancaster theory, thereby improving differentiation between a few highly mutated genes and many genes having only a few mutations apiece. We investigate accuracy, computational effort and power, reporting acceptable performance for each. Results: As an example calculation, we re-analyze KEGG-based lung adenocarcinoma pathway mutations from the Tumor Sequencing Project. Our test recapitulates the most significant pathways and finds that others for which the original test battery was inconclusive are not actually significant. It also identifies the focal adhesion pathway as being significantly mutated, a finding consistent with earlier studies. We also expand this analysis to other databases: Reactome, BioCarta, Pfam, PID and SMART, finding additional hits in ErbB and EPHA signaling pathways and regulation of telomerase. All have implications and plausible mechanistic roles in cancer. Finally, we discuss aspects of extending the method to integrate gene-specific background rates and other types of genetic anomalies. Availability: PathScan is implemented in Perl and is available from the Genome Institute at: http://genome.wustl.edu/software/pathscan. Contact: mwendl@wustl.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known.We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome.We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis.By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Using comparative sequencing approaches, we investigated the evolutionary history of the European-enriched 17q21.31 MAPT inversion polymorphism. We present a detailed, BAC-based sequence assembly of the inverted human H2 haplotype and contrast it with the sequence structure and genetic variation of the corresponding 1.5 Mb region for the non-inverted H1 human haplotype and that of chimpanzee and orangutan. We find that inversion of the MAPT region is similarly polymorphic in other great ape species and present evidence that the inversions have occurred independently in both chimpanzee and humans. In humans, the inversion breakpoints correspond to core duplications encoding the LRRC37 gene family. Our analysis favors the H2 configuration and sequence haplotype as the likely great ape/human ancestral state with inversion recurrences during primate evolution. We demonstrate that the H2 architecture has evolved more extensive sequence homology, perhaps explaining its preference to undergo microdeletion associated with mental retardation in European populations.

International audience; We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co- opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non- protein- coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

Here we present a draft genome sequence of the common chimpanzee (Pan troglodytes). Through comparison with the human genome, we have generated a largely complete catalogue of the genetic differences that have accumulated since the human and chimpanzee species diverged from our common ancestor, constituting approximately thirty-five million single-nucleotide changes, five million insertion/deletion events, and various chromosomal rearrangements. We use this catalogue to explore the magnitude and regional variation of mutational forces shaping these two genomes, and the strength of positive and negative selection acting on their genes. In particular, we find that the patterns of evolution in human and chimpanzee protein-coding genes are highly correlated and dominated by the fixation of neutral and slightly deleterious alleles. We also use the chimpanzee genome as an outgroup to investigate human population genetics and identify signatures of selective sweeps in recent human evolution.

Human chromosome 2 is unique to the human lineage in being the product of a head-to-head fusion of two intermediate-sized ancestral chromosomes. Chromosome 4 has received attention primarily related to the search for the Huntington's disease gene, but also for genes associated with Wolf-Hirschhorn syndrome, polycystic kidney disease and a form of muscular dystrophy. Here we present approximately 237 million base pairs of sequence for chromosome 2, and 186 million base pairs for chromosome 4, representing more than 99.6% of their euchromatic sequences. Our initial analyses have identified 1,346 protein-coding genes and 1,239 pseudogenes on chromosome 2, and 796 protein-coding genes and 778 pseudogenes on chromosome 4. Extensive analyses confirm the underlying construction of the sequence, and expand our understanding of the structure and evolution of mammalian chromosomes, including gene deserts, segmental duplications and highly variant regions.

As part of the effort to sequence the genome ofRattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide ∼13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing1, 2. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones3. In an accompanying paper4 we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes

Summary: Despite recent progress, computational tools that identify gene fusions from next-generation whole transcriptome sequencing data are often limited in accuracy and scalability. Here, we present a software package, BreakFusion that combines the strength of reference alignment followed by read-pair analysis and de novo assembly to achieve a good balance in sensitivity, specificity and computational efficiency. Availability: http://bioinformatics.mdanderson.org/main/BreakFusion Contact: kchen3@mdanderson.org; lding@genome.wustl.edu Supplementary information: Supplementary data are available at Bioinformatics online

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

Producing gene fusions through genomic structural rearrangements is a major mechanism for tumor evolution. Therefore, accurately detecting gene fusions and the originating rearrangements is of great importance for personalized cancer diagnosis and targeted therapy. We present a tool, BreakTrans, that systematically maps predicted gene fusions to structural rearrangements. Thus, BreakTrans not only validates both types of predictions, but also provides mechanistic interpretations. BreakTrans effectively validates known fusions and discovers novel events in a breast cancer cell line. Applying BreakTrans to 43 breast cancer samples in The Cancer Genome Atlas identifies 90 genomically validated gene fusions. BreakTrans is available at http://bioinformatics.mdanderson.org/main/BreakTrans.

Abstract 320 Multiple myeloma (MM) is an incurable malignancy of antibody secreting plasma B-cells whose etiology is still poorly understood. We used whole genome sequencing (WGS) and deep-read capture validation to thoroughly characterize the mutation landscape in four newly diagnosed MM patients and further examined mutation recurrence in 89 additional MM patients. WGS cases were selected to be racially diverse and represent both hyperdiploid and non-hyperdiploid MM with otherwise “simple” karyotypes. All studies to date have used peripheral blood as controls, however, abnormal B cells and circulating tumor cells frequently contaminate the peripheral blood of MM patients and these studies may have missed the early genetic events potentially important in disease pathogenesis. We therefore chose to use matched skin samples as normal controls. The use of skin DNA controls and deep read count capture validation of single nucleotide variants (SNV) allowed us, for the first time, to demonstrate clear separation between normal and malignant cell populations. Our analysis pipeline detected both somatic SNVs and structural variants (SV, ie. translocations, deletions, insertions). In all four patients, we observed chromosomal translocations at the Ig heavy chain locus, VDJ recombination, and Ig locus somatic hypermuation. We found somatic mutations affecting the E3 ubiquitin ligase HUWE1 in 4% (4/94) of cases, as well as recurring deletions affecting the Rho pathway regulator DIAPH2 (recurrence data will be presented). RNA sequencing was preformed for 3 of the WGS patients to explore transcriptional effects of mutations observed. Statistical analysis identified significantly mutated genes, i.e. mutation hotspots, including ROBO2, BCL6 and cadherin/catenin genes. Genes involved in cell polarity, cell adhesion and axon guidance were affected in all four WGS patients. Chromosomal translocations present in all four WGS patients involved both known oncogenes (CCND1, MYC and MAFB) and putative oncogenes. A novel translocation implicated KCNT2, encoding a sodium-activated potassium channel, as a potential oncogene in MM. Recurrent point mutations were relatively rare in MM, suggesting that SVs are more common driver mutations and/or that SNVs in MM disrupt a diverse array of genes to affect key pathways. For genomic studies in MM moving forward, our results emphasize the importance of matched normal controls uncontaminated by tumor cells, and suggest that careful analysis of SVs be included to find novel oncogenes and important clinical correlations. Disclosures: No relevant conflicts of interest to declare.

503 Background: To correlate clinical features of estrogen receptor positive breast cancer with somatic mutations, massively parallel sequencing (MPS) was applied to tumor and normal DNAs accrued from patients treated with neoadjuvant aromatase inhibitors (AI). Methods: MPS was applied to 77 baseline tumor biopsy samples from the preoperative letrozole trial (JACS 2009: 208, 906) and the Z1031 trial (JCO 2011: 29, 2342) followed by targeted sequencing in another 240 trial samples. Standard statistical approaches were used to compare mutation status and clinical parameters and pathway-based correlation was used to assess interactions between signaling perturbations induced by gene mutations and response to neoadjuvant AI. Results: Eighteen genes were significantly mutated above background. Aside from PIK3CA mutations, the list is dominated by loss-of-function mutations in tumor suppressor genes. Five (RUNX1, CBFP, MYH9, MLL3 and SF3B1) have been previously linked to benign and malignant hematopoietic disorders. Clinical correlation revealed that TP53 mutation was associated with PAM50 LumB status, high-grade histology and high proliferation rates whereas loss-of-function mutations in MAP3K1 associate with PAM50 lumA status, low proliferation rates, and low grade histology. Mutations in GATA3 were associated with greater suppression of proliferation upon AI treatment suggesting mutGATA3 may predict endocrine response. Notably, mutations in MAP3K1 were more common in PIK3CA mutant cases, suggesting cooperation. Pathway analysis demonstrated that rare MAP2K4 mutations produce similar pathway perturbations as MAP3K1 mutation, a logical finding since MAP2K4 is a substrate for MAP3K1. Signaling network patterns driven by lncRNA MALAT1 mutations were associated with multiple poor clinical outcome features. Rare mutations in druggable kinases included two in the kinase domain of HER2. Conclusions: Tumor heterogeneity in luminal-type breast cancer is driven by specific patterns of somatic mutations, however most druggable or potentially prognostic mutations are infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing approaches and large scale investigations.

Background: Estrogen receptors are over-expressed in around 70% of breast cancer cases. The genetic changes that occur during aromatase inhibitor (AI) treatment are not well understood and may differ depending upon the patient's response phenotype. Methods: We performed whole genome sequencing (WGS) of matched blood, pre-treatment, and post-treatment biopsy samples from 22 estrogen receptor positive breast cancer patients treated with neoadjuvant aromatase inhibitors. For 5 cases, we performed the whole genome sequencing (WGS) on patients’ matched normal, two pre AI-treatment, and two post AI-treatment DNA isolates from biopsy samples. We validated all putative coding and non-coding somatic mutations using deep sequencing. By comparing the validated somatic mutations from pre- and post- AI treatment biopsy samples, we were able to determine the alterations in the tumor genomes. In every case we defined the clonal architecture of each pair of pre-treatment and post-treatment biopsy samples by comparing the variant allele frequencies from thousands of validated somatic mutations. Results: Comparisons of the two pre AI-treatment biopsy samples from the same patient indicates that the variant allele frequencies of mutations showed high concordances in all 5 cases, 0.74 to 0.95 range of correlation coefficient. Only a small percentage of somatic mutations were detected in one pre-treatment sample and not the other (4.65% overall). In comparing the somatic variations between pre-treatment and matched post-treatment biopsy samples in 22 cases, we found that patients with good clinical response to AI treatment retained known driver mutations only in their pre-treatment tumors. Conversely, those patients with poor clinical response presented new driver mutations in their post-treatment samples. Furthermore, the variant allele frequency for most mutated genes decreased in post AI treatment samples for patients with good AI treatment response; on the contrary, the variant allele frequency increased for patients with poor clinical response. Conclusions: From WGS of matched normal, pre-treatment, and post-treatment biopsy samples, we identified new driver genes mutated in patients with poor clinical response, while patients with good clinical response had lost mutated driver genes in their post-treatment biopsy samples. The genetic landscape revealed by WGS of pre-treatment and post-treatment biopsy samples reveals mutational repertoires are remodeled by AI therapy. This finding suggests deep sequencing of AI treated samples will be necessary to reveal the complete complement of mutations present in a patient's tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-423. doi:1538-7445.AM2012-LB-423

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 microM. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S), adenylyl (beta,gamma-methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 micrograms/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.

Background: Endocrine-therapy resistant HER2 negative luminal-type breast cancer is the commonest cause of breast cancer death but the molecular basis for aggressive clinical behavior is poorly understood. Endocrine therapy resistant cases can be identified early in the course of the disease though the persistant expression of the cell cycle biomarker Ki67 despite neoadjuvant treatment with a potent aromatase inhibitor (JNCI 2008:100;159–61). Methods: Massively parallel DNA sequencing (Nature 2010; 464:999–1005) was applied to samples from two neoadjuvant endocrine trials (“POL” J Am Coll Surg. 2009; 208: 906–14 and “ACOSOG Z1031” JCO in press) to define the whole genome sequence of 50 luminal-type breast cancers (PAM50 definition, JCO 2009;27:1160–7) with an average 30 fold coverage. Of these, 24 were defined as resistant (surgical specimen Ki67 > 10%) and 26 sensitive (Ki67 ≤ 10%). Approximately 10 trillion base pairs of sequence were analyzed. Putative somatic mutations were confirmed through targeted sequence analysis and the absence of the variant in matched normal DNA. Results: The mean number of single nucleotide non-silent variants (SNV) and small deletions (indels) was greater in the resistant group (49 per case) than sensitive cases (23) (p=0.02). The significantly mutated gene list included PIK3CA, TP53, ATR, RUNX1, MYST3, PRSS8, ZNHIT2 and MAP3K1. A large number of structural variations have also been identified with greater than 800 events from 28 patients orthogonally validated to date, mostly deletions but also translocations, although no recurrent translocations have been seen. About 25% of these deletions were paired with an SNV suggesting multiple novel double-hit tumor suppressor events. Extension analysis in 121 POL/Z1031 samples produced the following mutation incidence data PIK3CA (43%), TP53 (15.2%) and MAP3K1 (9.3%). The MAP3K1 mutations were most frequently frame-shift or nonsense, disrupting the C terminal kinase domain, a logical finding in a kinase activated by caspase 7 to promote apoptosis. Only 1 MAP3K1 mutation was observed in 60 basal-like breast cancers (luminal versus basal MAP3K1 mutation frequency p=0.04). Conclusions: MAP3K1is a novel breast cancer tumor suppressor gene more frequently lost in luminal-type than basal-like breast cancer. Further data analysis will be presented including pathway analyses that place less common mutations into a variety of cancer pathways, including phosphoinositol-3-kinase signaling, cell cycle regulation, metabolism, mitotic spindle regulation and DNA mismatch repair. These analyses suggest a classification of the disease that is not based on individual gene abnormalities but common cancer pathway activation events that ultimately determine outcome for patients with luminal-type disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2011-LB-87

The ability to study genome-wide sequence alterations in cancer genomes has transformed our understanding of the intricacies of this disease. We have applied whole genome sequencing and analysis to tease apart the genomic predictors of aromatase inhibitor (AI) response, by selecting luminal breast cancer cases obtained within a clinical trial of aromatase inhibitor treatment (ACOSOG Z1031). Our study compared the constitutional genome to the cancer genome for patients that exhibit either a response or a resistance phenotype, based on their Ki67 IHC score following 4 month AI treatment and resection. My talk will highlight the process by which these genomes are sequenced and analyzed, and will provide our most recent insights into how the combination of the constitutional and somatic genomes determine whether a patient will respond to AI treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr ES7-1.

Abstract 2755 We describe a difficult diagnostic case of t(15;17)-negative acute promyelocytic leukemia (APL). A 39 year-old woman presented with pancytopenia and low grade DIC. Bone marrow biopsy revealed AML with promyelocytic features. She was treated with Cytarabine, Daunorubicin and ATRA. However, ATRA was discontinued after FISH revealed a possible RARA-PML, but no PML-RARA fusion (one fusion, one RARA and two PML signals), and cytogenetics did not demonstrate a translocation involving chromosome 15 or 17. In fact, her cytogenetics revealed a complex pattern that predicted poor prognosis (46 XX del(9)(q12q32),del(12)(q12q21)[6]/46,idem,-6,-16,add(16)(p13.2),+2 mar[13]/46 XX[1]). Following reinduction, she entered complete remission and was empirically consolidated with arsenic. RT-PCR for PML-RARA was not performed at diagnosis, and was negative at the time of consultation in remission. Her complex cytogenetics and uncertain FISH status posed a diagnostic and therapeutic dilemma that could only be resolved by whole genome sequencing in the time frame required for a clinical decision to be made (i.e. allogeneic transplantation vs. ATRA-based consolidation). DNA was therefore generated from bone marrow (cryopreserved at the time of diagnosis) and a skin sample (obtained in remission), and subjected to massively parallel sequencing using paired-end reads (Mardis et al, NEJM 2009). We generated 187 billion bp (tumor) and 200 billion bp (skin) of sequence, corresponding to 43.7x and 46.8x haploid coverage (99.76% and 99.74% diploid coverage) for the respective samples. Within six weeks of sample receipt, validated results were available. We confirmed the del(9)(q12q32) and del(12)(q12q21) somatic events and identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion: 77 Kb of chromosome 15 (chr15:72027045–72104108 containing LOXL1 exon 6 through PML exon 3) were inserted en bloc into RARA intron 2 (chr17:35742678–35742683). This event resulted in the expression of PML-RARA (bcr3 isoform) and two novel fusion transcripts (RARA-LOXL1 and LOXL1-PML), which were all successfully amplified by RT-PCR and sequenced. The RARA-LOXL1 and LOXL1-PML fusions both created stop codons shortly after the fusion events. Re-evaluation of the FISH results revealed that the insertion generated a fusion signal, while loss of 77 Kb from the PML locus did not prevent binding of the 239 Kb commercial PML probe to chromosome 15 (thus generating 1 fusion, 1 RARA and 2 PML signals). These signals represented the PML-RARA insertional fusion event, not the RARA-PML translocation that was originally reported. We further identified and validated deletions on chromosomes 12 (60 Mb), 14 (22 Kb) and 19 (30 Kb) and non-synonymous, somatic single nucleotide variants (SNVs) in the coding regions of ZNF687, DYTN, C3orf54, CH3D19, SLC35A4, GPRC6A, ZFHX4, PTK2, PITPNM1, DEGS2, PCSK2, CDC45L, although the clinical relevance of these deletions and point mutations is not yet known. After validating PML-RARA bcr3 expression, a decision was made to consolidate the patient with an ATRA-containing regimen. Using whole genome sequencing with paired end reads, we have identified a novel oncogenic form of chromosomal rearrangement, an insertional fusion. Similar insertional events may occur in other loci. Small structural events (under a few megabases in size) are often undetectable by conventional cytogenetics and FISH, and are expected to be invisible to standard break-apart probes (commonly used to evaluate the RARA and MYC loci). This case highlights the clinical relevance of whole genome sequencing for informing diagnostic and therapeutic decisions that must be made within weeks after sample acquisition. Disclosures: Off Label Use: Decitabine, Arsenic and Ascorbic acid for the treatment of AML. DiPersio:Genzyme: Honoraria. Westervelt:Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau.

10500 Background: Massively parallel DNA sequencing technology facilitates new approaches for unbiased characterization of cancer genomes. We have pursued a novel strategy to identify mutations tha...

The two genetically unlinked histone H2B genes isolated from Saccharomyces cerevisiae have been sequenced. The genes encode H2B proteins that are 130 amino acids in length and that differ by 4 amino acids. The changes betwen them are Ala leads to Ser, Lys leads to Ala, Thr leads to Val and Ala leads to Val at amino acid positions 2, 3, 27 and 35, respectively. A comparison of yeast H2B histones with those of higher eucaryotes demonstrates a high degree of homology clustered mainly at the carboxyl terminus. There is extensive base substitution between the two H2B genes in nucleotides that do not affect the amino acid sequence. DNA prelude sequences show 64% divergence. The coding regions differ at 49 of the 390 bases (12.6% divergence). 41 of these changes are in silent positions. By using the number of amino acid differences in the proteins we estimate that the two H2B genes are the result of an ancient duplication event.

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.

Saccharomyces cerevisiae contains two histone H2B protein subtypes, H2B1 and H2B2, which differ at 4 of 130 amino acids. We describe experiments that test whether both histone H2B subtypes are required for the completion of any stage in the yeast life cycle. Frameshift mutations were introduced into cloned copies of the H2B1 and H2B2 genes. These altered genes were integrated into the yeast genome by transformation and replaced the wild-type genes through recombination. We thus obtained strains that lacked functional H2B1 or H2B2 proteins. These mutant strains survive as haploids and homozygous diploids. During vegetative growth, they divide at the same rate as wild-type cells and are able to mate, sporulate and germinate. The h2b1 − cells grew more slowly after germination than h2b2 − or wild-type spores, but otherwise the mutants were indistinguishable from each other or from wild-type cells. We also attempted to make a strain that was mutant in both genes for H2B. We examined spores derived from a diploid that is heterozygous for both histone mutations. The two genes assort independently, so we expect one in four spores to be h2b1 − h2b2 − . Of 61 spore colonies examined, none was mutant at both loci. Our results indicate that the double mutant can germinate and bud once but cannot grow further. Since the yeast life cycle can be completed in the absence of either but not both histone H2B subtypes, we conclude that neither protein has a unique essential function.

The basic amino terminus of each histone is external to the nucleosome core particle. In vitro studies have shown that the amino termini are not required for nucleosome assembly. To address the significance of these results in vivo we constructed mutations in yeast histone H2B in genetic backgrounds lacking wild-type H2B protein. We found that the protein can function in vivo even with large deletions at its amino terminus. These mutations produce no obvious phenotype and there appears to be no selection against the mutant proteins in chromatin assembly. A deletion removing a large portion of the carboxyl terminus was lethal. We conclude that much of the amino terminus of histone H2B has no essential function in vivo.