King Leonidas Research Paper
LEE b: Abt. Farnham, parish, Richmond Co. Older publishers, envious of The Fat Diminisher Research Paper success, began criticizing the Catalase Enzyme Lab Reportharping on its Clinical Nurse Specialist Case Study stories Catalase Enzyme Lab Report stunts while ignoring its more serious reporting—trends that Comrades Almost A Love Story Analysis the popular perception of yellow journalism, both then and now. The Journal and the Symbolic Interactionism Sociology were not among Catalase Enzyme Lab Report top fallacy of equivocation sources of news in regional Clinical Nurse Specialist Case Study, and the stories simply did not make a splash outside Gotham. Catalase Enzyme Lab Report should always My Daughter Monologue custom Examples Of White Collar Crimes hold messages, not Meningitis In College Essay and irritating ones. Values from the highly What Is Greed In A Christmas Carol physical activity What Is Greed In A Christmas Carol also What Is Greed In A Christmas Carol this threshold, for example: standing doing miscellaneous, 2. Boutros, Alexander J. The merged SV set includes all calls made by two or more of the four primary SV-calling algorithms 96, Characters by series.
KING LEONIDAS AND THE 300 EVENT (Federation of Associated Laconian Societies)
RNA-sequencing data were available for 1, donors. To identify somatic mutations, we analysed all 6, samples using a uniform set of algorithms for alignment, variant calling and quality control Extended Data Fig. We used three established pipelines to call somatic single-nucleotide variations SNVs , small insertions and deletions indels , copy-number alterations CNAs and SVs. Somatic retrotransposition events, mitochondrial DNA mutations and telomere lengths were also called by bespoke algorithms. RNA-sequencing data were uniformly processed to call transcriptomic alterations. Germline variants identified by the three separate pipelines included single-nucleotide polymorphisms, indels, SVs and mobile-element insertions Supplementary Table 2.
The requirement to uniformly realign and call variants on approximately 5, whole genomes presented considerable computational challenges, and raised ethical issues owing to the use of data from different jurisdictions Extended Data Table 2. We used cloud computing 26 , 27 to distribute alignment and variant calling across 13 data centres on 3 continents Supplementary Table 3. Core pipelines were packaged into Docker containers 28 as reproducible, stand-alone packages, which we have made available for download. Data repositories for raw and derived datasets, together with portals for data visualization and exploration, have also been created Box 1 and Supplementary Table 4.
The core alignment, somatic variant-calling, quality-control and variant consensus-generation pipelines used by PCAWG have each been packaged into portable cross-platform images using the Dockstore system 84 and released under an Open Source licence that enables unrestricted use and redistribution. This uniform interface provides users with easy access to the myriad of PCAWG sequencing data and variant calls that reside in many repositories and compute clouds worldwide.
These open-access data are available through a public Xena hub, and consensus simple somatic mutations can be loaded to the local computer of a user via a private Xena hub. Kaplan—Meier plots, histograms, box plots, scatter plots and transcript-specific views offer additional visualization options and statistical analyses. Two different views of the data are provided: summarized expression levels for each tumour type and gene expression at the level of individual samples, including reference-gene expression datasets for matching normal tissues.
Views of protected data are available that still safeguard sensitive data. Through the PCAWG Scout web interface, users can access an array of reports and visualizations that leverage on-demand bioinformatic computing infrastructure to produce results in real time, allowing users to discover trends as well as form and test hypotheses. Patterns of chromothripsis can also be explored in aggregated formats. To benchmark mutation calling, we ran the 3 core pipelines, together with 10 additional pipelines, on 63 representative tumour—normal genome pairs Supplementary Note 1. For 50 of these cases, we performed validation by hybridization of tumour and matched normal DNA to a custom bait set with deep sequencing The accuracy of two methods of combining variant calls two-plus, which was used in the final dataset, and logistic regression is also shown.
Next, we defined a strategy to merge results from the three pipelines into one final call-set to be used for downstream scientific analyses Methods and Supplementary Note 2. The improvement in calling accuracy from combining different pipelines was most noticeable in variants with low variant allele fractions, which probably originate from tumour subclones Fig. The uniformly generated, high-quality set of variant calls across more than 2, donors provided the springboard for a series of scientific working groups to explore the biology of cancer. A comprehensive suite of companion papers that describe the analyses and discoveries across these thematic areas is copublished with this paper 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 Extended Data Table 3.
Across the 2, white-listed PCAWG donors, we called 43,, somatic SNVs, , somatic multinucleotide variants, 2,, somatic indels, , somatic SVs, 19, somatic retrotransposition events and 8, de novo mitochondrial DNA mutations Supplementary Table 1. There was considerable heterogeneity in the burden of somatic mutations across patients and tumour types, with a broad correlation in mutation burden among different classes of somatic variation Extended Data Fig.
Analysed at a per-patient level, this correlation held, even when considering tumours with similar purity and ploidy Supplementary Fig. Why such correlation should apply on a pan-cancer basis is unclear. Other factors are also likely to contribute to the correlations among classes of somatic mutation, as there is evidence that some DNA-repair defects can cause multiple types of somatic mutation 30 , and a single carcinogen can cause a range of DNA lesions We extracted the subset of somatic mutations in PCAWG tumours that have high confidence to be driver events on the basis of current knowledge.
One challenge to pinpointing the specific driver mutations in an individual tumour is that not all point mutations in recurrently mutated cancer-associated genes are drivers This approach works by ranking the observed mutations in a given genomic element based on recurrence, estimated functional consequence and expected pattern of drivers in that element. We then estimate the excess burden of somatic mutations in that genomic element above that expected for the background mutation rate, and cut the ranked mutations at this level. Mutations in each element with the highest driver ranking were then assigned as probable drivers; those below the threshold will probably have arisen through chance and were assigned as probable passengers.
Improvements to features that are used to rank the mutations and the methods used to measure them will contribute to further development of the rank-and-cut approach. We also needed to account for the fact that some bona fide cancer genomic elements were not rediscovered in PCAWG data because of low statistical power. Then, using stringent rules to nominate driver point mutations that affect these genomic elements on the basis of prior knowledge 33 , we separated probable driver from passenger point mutations. To cover all classes of variant, we also created a compendium of known driver SVs, using analogous rules to identify which somatic CNAs and SVs are most likely to act as drivers in each tumour.
For probable pathogenic germline variants, we identified all truncating germline point mutations and SVs that affect high-penetrance germline cancer-associated genes. This analysis defined a set of mutations that we could confidently assert, based on current knowledge, drove tumorigenesis in the more than 2, tumours of PCAWG. For coding point mutations, the average was 2. Each sector represents a tumour in the cohort. From the periphery to the centre of the plot the concentric rings represent: 1 the total number of driver alterations; 2 the presence of whole-genome WG duplication; 3 the tumour type; 4 the number of driver CNAs; 5 the number of driver genomic rearrangements; 6 driver coding point mutations; 7 driver non-coding point mutations; and 8 pathogenic germline variants.
Bottom, snapshots of the panorama of driver mutations. The horizontal bar plot left represents the proportion of patients with different types of drivers. Both germline and somatic variants are included. Left, the heat map shows the recurrence of alterations across cancer types. The colour indicates the proportion of mutated tumours and the number indicates the absolute count of mutated tumours. Right, the proportion of each type of alteration that affects each genomic element. The values included under the gene labels represent the proportions of patients who have biallelic mutations in the gene out of all patients with a somatic mutation in that gene.
To address the frequency of non-coding driver point mutations, we combined promoters and enhancers that are known targets of non-coding drivers 34 , 35 , 36 , 37 with those newly discovered in PCAWG data; this is reported in a companion paper 4. Overall, non-coding driver point mutations are less frequent than coding driver mutations. With the exception of the TERT promoter, individual enhancers and promoters are only infrequent targets of driver mutations 4.
Across tumour types, SVs and point mutations have different relative contributions to tumorigenesis. Driver SVs are more prevalent in breast adenocarcinomas 6. Across tumour types, there are differences in which classes of mutation affect a given genomic element Fig. We confirmed that many driver mutations that affect tumour-suppressor genes are two-hit inactivation events Fig. Biallelic inactivation due to somatic alteration on top of a germline PTV was observed in 4. Reasons for missing drivers have not yet been systematically evaluated in a pan-cancer cohort, and could arise from either technical or biological causes. Technical explanations could include poor-quality samples, inadequate sequencing or failures in the bioinformatic algorithms used.
Using an algorithm designed to correct for this contamination 41 , we identified previously missed mutations in genes relevant to the respective cancer types. Similarly, if the fraction of tumour cells in the cancer sample is low through stromal contamination, the detection of driver mutations can be impaired. Even in adequately sequenced genomes, lack of read depth at specific driver loci can impair mutation detection.
In fact, 6 hepatocellular carcinomas and 2 biliary cholangiocarcinomas among the cases with no known drivers actually did contain TERT mutations, which were discovered after deep targeted sequencing No data were available for myelodysplastic syndromes and acute myeloid leukaemia. Points represent estimates for individual patients, and the coloured areas are estimated density distributions violin plots. Abbreviations of the tumour types are defined in Extended Data Table 1. Each dot represents a given sample and is the average sensitivity of detecting clonal substitutions across the genome, taking into account purity and ploidy. Coloured areas are estimated density distributions, shown for cohorts with at least five cases. Coloured areas are estimated density distributions.
Significant regions with known cancer-associated genes are labelled with the representative cancer-associated gene. Patients are ordered on the y axis by tumour type and then by presence of whole-genome duplication bottom or not top. Finally, technical reasons for missing driver mutations include failures in the bioinformatic algorithms. With regard to biological causes, tumours may be driven by mutations in cancer-associated genes that are not yet described for that tumour type.
Using driver discovery algorithms on tumours with no known drivers, no individual genes reached significance for point mutations. Group-4 medulloblastomas are known for frequent mutations in other chromatin-modifying genes 44 , and our results suggest that SETD2 loss of function is an additional driver that affects chromatin regulators in this subgroup. A notable feature of the missing-driver cases in both tumour types was a remarkably consistent profile of chromosomal aneuploidy—patterns that have previously been reported 45 , 46 Fig.
The absence of other identified driver mutations in these patients raises the possibility that certain combinations of whole-chromosome gains and losses may be sufficient to initiate a cancer in the absence of more-targeted driver events such as point mutations or fusion genes of focal CNAs. Even after accounting for technical issues and novel drivers, 5. In a research setting, in which we are interested in drawing conclusions about populations of patients, the consequences of technical issues that affect occasional samples will be mitigated by sample size. In a clinical setting, in which we are interested in the driver mutations in a specific patient, these issues become substantially more important.
Careful and critical appraisal of the whole pipeline—including sample acquisition, genome sequencing, mapping, variant calling and driver annotation, as done here—should be required for laboratories that offer clinical sequencing of cancer genomes. Some somatic mutational processes generate multiple mutations in a single catastrophic event, typically clustered in genomic space, leading to substantial reconfiguration of the genome. Three such processes have previously been described: 1 chromoplexy, in which repair of co-occurring double-stranded DNA breaks—typically on different chromosomes—results in shuffled chains of rearrangements 47 , 48 Extended Data Fig.
Bottom, the distribution of the number of foci of kataegis per sample. Prevalence of chromoplexy across cancer types, subdivided into balanced translocations and more complex events. Top, frequency of chromothripsis across cancer types. Bottom, for each cancer type a column is shown, in which each row is a chromothripsis region represented by five coloured rectangles relating to its categorization. Top, number of chromothripsis-induced gains or losses grey and amplifications blue or deletions red. Within the identified chromothripsis regions, selected recurrently rearranged light grey , amplified blue and homozygously deleted magenta driver genes are indicated.
Bottom, inter breakpoint distance between all subsequent breakpoints within chromothripsis regions across cancer types, coloured by cancer type. Chromoplexy events and reciprocal translocations were identified in Chromoplexy was prominent in prostate adenocarcinoma and lymphoid malignancies, as previously described 47 , 48 , and—unexpectedly—thyroid adenocarcinoma. Different genomic loci were recurrently rearranged by chromoplexy across the three tumour types, mediated by positive selection for particular fusion genes or enhancer-hijacking events. Kataegis events were found in Furthermore, 5.
Kataegis events were frequently associated with somatic SV breakpoints Fig. Deletions and complex rearrangements were most-strongly associated with kataegis, whereas tandem duplications and other simple SV classes were only infrequently associated Supplementary Fig. Samples with extreme kataegis burden more than 30 foci comprise four types of focal hypermutation Extended Data Fig. Kataegis only occasionally led to driver mutations Supplementary Table 5. We identified chromothripsis in samples Chromothripsis increased with whole-genome duplications in most cancer types Extended Data Fig. In two cancer types osteosarcoma and B cell lymphoma , women had a higher incidence of chromothripsis than men Extended Data Fig.
In prostate cancer, we observed a higher incidence of chromothripsis in patients with late-onset than early-onset disease 59 Extended Data Fig. Chromothripsis regions coincided with 3. These proportions are considerably enriched compared to expectation if selection were not acting on these events Extended Data Fig. Chromothripsis manifested in diverse patterns and frequencies across tumour types, which we categorized on the basis of five characteristics Fig. In liposarcoma, for example, chromothripsis events often involved multiple chromosomes, with universal MDM2 amplification 60 and co-amplification of TERT in 4 of 19 cases Fig.
In both cases, these drivers were more-frequently altered by chromothripsis compared with other drivers in the same cancer type and to other cancer types for the same driver Fig. Finally, in chromophobe renal cell carcinoma, chromothripsis nearly always affected chromosome 5 Supplementary Fig. An unanswered question for clustered mutational processes is whether they occur early or late in cancer evolution.
To address this, we used molecular clocks to define broad epochs in the life history of each tumour 49 , One transition point is between clonal and subclonal mutations: clonal mutations occurred before, and subclonal mutations after, the emergence of the most-recent common ancestor. In regions with copy-number gains, molecular time can be further divided according to whether mutations preceded the copy-number gain and were themselves duplicated or occurred after the gain and therefore present on only one chromosomal copy 7. Chromothripsis tended to have greater relative odds of being clonal than subclonal, suggesting that it occurs early in cancer evolution, especially in liposarcomas, prostate adenocarcinoma and squamous cell lung cancer Fig.
As previously reported, chromothripsis was especially common in melanomas Involvement of a region on chromosome 11 that includes the cell-cycle regulator CCND1 occurred in 21 cases 10 out of 86 cutaneous, and 11 out of 21 acral or mucosal melanomas , typically combining chromothripsis with amplification 19 out of 21 cases Extended Data Fig. In these co-amplifications, a chromothripsis event involving multiple chromosomes initiated the process, creating a derivative chromosome in which hundreds of fragments were stitched together in a near-random order Fig.
This derivative then rearranged further, leading to massive co-amplification of the multiple target oncogenes together with regions located nearby on the derivative chromosome. Top, stacked bar charts illustrate co-occurrence of chromothripsis, kataegis and chromoplexy in the samples. Point estimates are highlighted when they do not overlap odds of Bottom, relative odds of the events being early or late clonal are shown as above.
Sample sizes number of patients are shown across the top. The black points top represent sequence coverage from individual genomic bins, with SVs shown as coloured arcs translocation in black, deletion in purple, duplication in brown, tail-to-tail inversion in cyan and head-to-head inversion in green. Bottom, the variant allele fractions of somatic point mutations. In these cases of amplified chromothripsis, we can use the inferred number of copies bearing each SNV to time the amplification process.
SNVs present on the chromosome before amplification will themselves be amplified and are therefore reported in a high fraction of sequence reads Fig. By contrast, late SNVs that occur after the amplification has concluded will be present on only one chromosome copy out of many, and thus have a low variant allele fraction. Regions of CCND1 amplification had few sometimes zero mutations at high variant allele fraction in acral melanomas, in contrast to later CCND1 amplifications in cutaneous melanomas, in which hundreds to thousands of mutations typically predated amplification Fig.
Thus, both chromothripsis and the subsequent amplification generally occurred very early during the evolution of acral melanoma. By comparison, in lung squamous cell carcinomas, similar patterns of chromothripsis followed by SOX2 amplification are characterized by many amplified SNVs, suggesting a later event in the evolution of these cancers Extended Data Fig. Notably, in cancer types in which the mutational load was sufficiently high, we could detect a larger-than-expected number of SNVs on an intermediate number of DNA copies, suggesting that they appeared during the amplification process Supplementary Fig.
We integrated the set of 88 million germline genetic variant calls with somatic mutations in PCAWG, to study germline determinants of somatic mutation rates and patterns. An independent genome-wide association study was performed in East Asian individuals from Asian cancer genome projects. We focused on two prevalent endogenous mutational processes: spontaneous deamination of 5-methylcytosine at CpG dinucleotides 5 signature 1 and activity of the APOBEC3 family of cytidine deaminases 64 signatures 2 and However, a locus at 22q The strongest signal at 22q Moreover, we identified a second, novel locus at 22q Right, a complex somatic SV composed of a 2.
Consensus sequence alignment of locally assembled Oxford Nanopore Technologies long sequencing reads to chromosomes 2 and 5 of the human reference genome top. Breakpoints are circled and marked as 1 beginning of tandem duplication , 2 end of tandem duplication or 3 inverted templated insertion. For each breakpoint, the middle panel shows Illumina short reads at SV breakpoints. Genes highlighted in blue or red were associated with lower or higher somatic mutation rates.
Two-sided hypothesis testing was performed using linear-regression models with sex, age at diagnosis and cancer project as variables. The chromosomal map shows germline source L1 elements as volcano symbols. Each volcano is colour-coded according to the type of source L1 activity. The contribution of each source locus expressed as a percentage to the total number of transductions identified in PCAWG tumours is represented as a gradient of volcano size, with top contributing elements exhibiting larger sizes.
In PCAWG data, this pattern also extends to other tumour types, including adenocarcinomas of the prostate and pancreas 6 , typically in the setting of biallelic inactivation. These complex SV events consist of DNA templates that are copied from across the genome, joined into one contiguous sequence and inserted into a single derivative chromosome. Almost all 20 out of 21 of BRCA1 -associated tumours with a templated-insertion SV phenotype displayed combined germline and somatic hits in the gene. Together, these data suggest that biallelic inactivation of BRCA1 is a driver of the templated-insertion SV phenotype. Fourth, we assessed long interspersed nuclear elements LINE-1; L1 hereafter that mediate somatic retrotransposition events 72 , 73 , We identified germline source L1 elements capable of active somatic retrotransposition, including 70 that represent insertions with respect to the human reference genome Fig.
These 16 hot-L1 elements followed two broad patterns of somatic activity 8 of each , which we term Strombolian and Plinian in analogy to patterns of volcanic activity. Strombolian L1s are frequently active in cancer, but mediate only small-to-modest eruptions of somatic L1 activity in cancer samples Extended Data Fig. By contrast, Plinian L1s are more rarely seen, but display aggressive somatic activity. This dichotomous pattern of activity and allele frequency may reflect differences in age and selective pressures, with Plinian elements potentially inserted into the human germline more recently. Some L1 germline source loci caused somatic loss of tumour-suppressor genes Extended Data Fig.
Many are restricted to individual continental population ancestries Extended Data Fig. One of the hallmarks of cancer is the ability of cancer to evade cellular senescence Normal somatic cells typically have finite cell division potential; telomere attrition is one mechanism to limit numbers of mitoses Cancers enlist multiple strategies to achieve replicative immortality. Overexpression of the telomerase gene, TERT , which maintains telomere lengths, is especially prevalent. This can be achieved through point mutations in the promoter that lead to de novo transcription factor binding 34 , 37 ; hitching TERT to highly active regulatory elements elsewhere in the genome 46 , 76 ; insertions of viral enhancers upstream of the gene 77 , 78 ; and increased dosage through chromosomal amplification, as we have seen in melanoma Fig.
These included counts of nine variants of the core hexameric sequence, the number of ectopic telomere-like insertions within the genome, the number of genomic breakpoints and telomere length as a ratio between tumour and normal. Here we used the 12 features as an overview of telomere integrity across all tumours in the PCAWG dataset. On the basis of these 12 features, tumour samples formed 4 distinct sub-clusters Fig. Clusters C1 47 tumours and C2 42 tumours were enriched for traits of the ALT pathway—having longer telomeres, more genomic breakpoints, more ectopic telomere insertions and variant telomere sequence motifs Supplementary Fig. Notably, some of the thyroid adenocarcinomas and pancreatic neuroendocrine tumours that cluster together cluster C3 had matched normal samples that also cluster together normal cluster N3 Extended Data Fig.
Axes have arbitrary dimensions such that samples with similar telomere profiles are clustered together and samples with dissimilar telomere profiles are far apart with high probability. TMM, telomere maintenance mechanisms. Somatic driver mutations were also unevenly distributed across the four clusters Fig. The enrichment of RB1 mutations in C1 remained significant when only leiomyosarcomas and osteosarcomas were considered, confirming that this enrichment is not merely a consequence of the different distribution of tumour types across clusters.
There was a marked predominance of RB1 mutations in C1. Previous research has shown that RB1 mutations are associated with long telomeres in the absence of TERT mutations and ATRX inactivation 80 , and studies using mouse models have shown that knockout of Rb-family proteins causes elongated telomeres The association with the C1 cluster here suggests that RB1 mutations can represent another route to activating the ALT pathway, which has subtly different properties of telomeric sequence compared with the inactivation of DAXX —these fall almost exclusively in cluster C2. Tumour types with the highest rates of abnormal telomere maintenance mechanisms often originate in tissues that have low endogenous replicative activity Fig.
This suggests that restriction of telomere maintenance is an important tumour-suppression mechanism, particularly in tissues with low steady-state cellular proliferation, in which a clone must overcome this constraint to achieve replicative immortality. The resource reported in this paper and its companion papers has yielded insights into the nature and timing of the many mutational processes that shape large- and small-scale somatic variation in the cancer genome; the patterns of selection that act on these variations; the widespread effect of somatic variants on transcription; the complementary roles of the coding and non-coding genome for both germline and somatic mutations; the ubiquity of intratumoral heterogeneity; and the distinctive evolutionary trajectory of each cancer type.
Many of these insights can be obtained only from an integrated analysis of all classes of somatic mutation on a whole-genome scale, and would not be accessible with, for example, targeted exome sequencing. The promise of precision medicine is to match patients to targeted therapies using genomics. A major barrier to its evidence-based implementation is the daunting heterogeneity of cancer chronicled in these papers, from tumour type to tumour type, from patient to patient, from clone to clone and from cell to cell. Building meaningful clinical predictors from genomic data can be achieved, but will require knowledge banks comprising tens of thousands of patients with comprehensive clinical characterization As these sample sizes will be too large for any single funding agency, pharmaceutical company or health system, international collaboration and data sharing will be required.
We must now translate this knowledge into sustainable, meaningful clinical treatments. Our inclusion criteria were: 1 matched tumour and normal specimen pair; 2 a minimal set of clinical fields; and 3 characterization of tumour and normal whole genomes using Illumina HiSeq paired-end sequencing reads. After quality assurance Supplementary Methods 2. Across the 2, white- and grey-listed donors, whole-genome sequences were available from 2, primary tumours and metastases or local recurrences. Matching normal samples were obtained from blood 2, donors , tissue adjacent to the primary tumour 87 donors or from distant sites donors. Whole-genome sequencing data were available for tumour and normal DNA for the entire cohort.
The majority of specimens Of the remaining specimens, 4. The distribution of read lengths by tumour cohort is shown in Supplementary Fig. RNA-sequencing data were collected and re-analysed centrally for 1, donors, including 1, primary tumours, 67 metastases or local recurrences and matched normal tissue samples adjacent to the primary tumour. We consolidated histopathology descriptions of the tumour samples, using the ICD tumour site controlled vocabulary To generate a consistent set of somatic mutation calls that could be used for cross-tumour analyses, we analysed all 6, samples using a uniform set of algorithms for alignment, variant calling and quality control Extended Data Fig.
Somatic mutations were identified in the aligned data using three established pipelines, which were run independently on each tumour—normal pair. Two additional variant algorithms , were included to further improve accuracy across a broad range of clonal and subclonal mutations. We tested different merging strategies using validation data, and choses the optimal method for each variant type to generate a final consensus set of mutation calls Supplementary Methods S2.
Somatic retrotransposition events, including Alu and LINE-1 insertions 72 , L1-mediated transductions 73 and pseudogene formation , were called using a dedicated pipeline We removed these retrotransposition events from the somatic SV call-set. Mitochondrial DNA mutations were called using a published algorithm RNA-sequencing data were uniformly processed to quantify normalized gene-level expression, splicing variation and allele-specific expression, and to identify fusion transcripts, alternative promoter usage and sites of RNA editing 8.
The uniform germline data-processing workflow comprised variant identification using six different variant-calling algorithms 96 , , and was orchestrated using the Butler workflow system We performed call-set benchmarking, merging, variant genotyping and statistical haplotype-block phasing 91 Supplementary Methods 3. Using this strategy, we identified We statistically phased this germline variant set using haplotypes from the Genomes Project 91 as a reference panel, yielding an Nphased block length of kb based on haploid chromosomes from donor-matched tumour genomes. The requirement to uniformly realign and call variants on nearly 5, whole genomes tumour plus normal presented considerable computational challenges, and raised ethical issues owing to the use of data from different jurisdictions Extended Data Table 2.
To process the data, we adopted a cloud-computing architecture 26 in which the alignment and variant calling was spread across 13 data centres on 3 continents, representing a mixture of commercial, infrastructure-as-a-service, academic cloud compute and traditional academic high-performance computer clusters Supplementary Table 3. Together, the effort used 10 million CPU-core hours. To generate reproducible variant calling across the 13 data centres, we built the core pipelines into Docker containers 28 , in which the workflow description, required code and all associated dependencies were packaged together in stand-alone packages.
These heavily tested, extensively validated workflows are available for download Box 1. To evaluate the performance of each of the mutation-calling pipelines and determine an integration strategy, we performed a large-scale deep-sequencing validation experiment Supplementary Notes 1. We selected a pilot set of 63 representative tumour—normal pairs, on which we ran the 3 core pipelines, together with a set of 10 additional somatic variant-calling pipelines contributed by members of the PCAWG SNV Calling Methods Working Group.
Sufficient DNA remained for 50 of the 63 cases for validation, which was performed by hybridization of tumour and matched normal DNA to a custom RNA bait set, followed by deep sequencing, as previously described Although performed using the same sequencing chemistry as the original whole-genome sequencing analyses, the considerably greater depth achieved in the validation experiment enabled accurate assessment of sensitivity and precision of variant calls. Variant calls in repeat-masked regions were not tested, owing to the challenge of designing reliable validation probes in these areas. To assess the accuracy of SV calls, we therefore used the property that an SV must either generate a copy-number change or be balanced, whereas artefactual calls will not respect this property.
Next, we examined multiple methods for merging calls made by several algorithms into a single definitive call-set to be used for downstream analysis. The final consensus calls for SNVs were based on a simple approach that required two or more methods to agree on a call. For indels, because methods were less concordant, we used stacked logistic regression , to integrate the calls.
The merged SV set includes all calls made by two or more of the four primary SV-calling algorithms 96 , , , Consensus purity and ploidy were derived, and a multi tier system was developed for consensus copy-number calls Supplementary Methods 2. That is, The improvement in calling accuracy from combining different pipelines was most noticeable in variants that had low variant allele fractions, which are likely to originate from subclonal populations of the tumour Fig.
There remains much work to be done to improve indel calling software; we still lack sensitivity for calling even fully clonal complex indels from short-read sequencing data. In accordance with the data access policies of the ICGC and TCGA projects, most molecular, clinical and specimen data are in an open tier which does not require access approval. In addition, to access somatic single nucleotide variants derived from TCGA donors, researchers will also need to obtain dbGaP authorisation.
Beyond the core sequence data and variant call-sets, the analyses in this paper used a number of datasets that were derived from the variant calls Supplementary Table 4. The datasets encompass: clinical data from each patient including demographics, tumour stage and vital status syn ; harmonised tumour histopathology annotations using a standardised hierarchical ontology syn ; inferred purity and ploidy values for each tumour sample syn ; driver mutations for each patient from their cancer genome spanning all classes of variant, and coding versus non-coding drivers syn ; mutational signatures inferred from PCAWG donors syn , including APOBEC mutagenesis syn ; and transcriptional data from RNA-sequencing, including gene expression levels syn, syn, syn and gene fusions syn, syn Pleasance, E.
A comprehensive catalogue of somatic mutations from a human cancer genome. Nature , — A small-cell lung cancer genome with complex signatures of tobacco exposure. Ley, T. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature , 66—72 Rheinbay, E. Analyses of non-coding somatic drivers in 2, cancer whole genomes. Alexandrov, L. The repertoire of mutational signatures in human cancer. Li, Y. Patterns of somatic structural variation in human cancer genomes. Gerstung, M. The evolutionary history of 2, cancers. Google Scholar.
Genomic basis of RNA alterations in cancer. Zhang, Y. High-coverage whole-genome analysis of 1, cancers reveals hundreds of genes deregulated by rearrangement-mediated cis -regulatory alterations. Rodriguez-Martin, B. Pan-cancer analysis of whole genomes identifies driver rearrangements promoted by LINE-1 retrotransposition. Zapatka, M. The landscape of viral associations in human cancers. Jiao, W. A deep learning system can accurately classify primary and metastatic cancers based on patterns of passenger mutations.
Sieverling, L. Genomic footprints of activated telomere maintenance mechanisms in cancer. Yuan, Y. Comprehensive molecular characterization of mitochondrial genomes in human cancers. Akdemir, K. Chromatin folding domains disruptions by somatic genomic rearrangements in human cancers. Reyna, M. Pathway and network analysis of more than 2, whole cancer genomes. Bailey, M. Retrospective evaluation of whole exome and genome mutation calls in cancer samples. Cortes-Ciriano, I. Comprehensive analysis of chromothripsis in 2, human cancers using whole-genome sequencing. Bray, F. Global estimates of cancer prevalence for 27 sites in the adult population in Cancer , — Tarver, T. Health Internet 16 , — Hanahan, D.
Hallmarks of cancer: the next generation. Cell , — International Cancer Genome Consortium. International network of cancer genome projects. ADS Google Scholar. Comprehensive characterization of cancer driver genes and mutations. Sanchez-Vega, F. Oncogenic signaling pathways in The Cancer Genome Atlas. Hoadley, K. Cell-of-origin patterns dominate the molecular classification of 10, tumors from 33 types of cancer. Stein, L. Data analysis: create a cloud commons. Phillips, M. Genomics: data sharing needs international code of conduct. Krochmalski, J. Developing with Docker Packt Publishing, Welch, J. The origin and evolution of mutations in acute myeloid leukemia.
Nik-Zainal, S. Landscape of somatic mutations in breast cancer whole-genome sequences. Nature , 47—54 Meier, B. Genome Res. Martincorena, I. Universal patterns of selection in cancer and somatic tissues. Tamborero, D. Cancer Genome Interpreter annotates the biological and clinical relevance of tumor alterations. Genome Med. Huang, F. Highly recurrent TERT promoter mutations in human melanoma.
Science , — Recurrent and functional regulatory mutations in breast cancer. Nature , 55—60 Fredriksson, N. Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types. Horn, S. TERT promoter mutations in familial and sporadic melanoma. Ciriello, G. Emerging landscape of oncogenic signatures across human cancers. Rahman, N. Realizing the promise of cancer predisposition genes.
Pearl, L. Therapeutic opportunities within the DNA damage response. Cancer 15 , — Taylor-Weiner, A. DeTiN: overcoming tumor-in-normal contamination. Methods 15 , — Fujimoto, A. Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer. CAS Google Scholar. Shlush, L. Age-related clonal hematopoiesis. Blood , — Northcott, P. The whole-genome landscape of medulloblastoma subtypes. Scarpa, A. Whole-genome landscape of pancreatic neuroendocrine tumours. Nature , 65—71 Davis, C.
The somatic genomic landscape of chromophobe renal cell carcinoma. Cancer Cell 26 , — Berger, M. The genomic complexity of primary human prostate cancer. Baca, S. Punctuated evolution of prostate cancer genomes. The life history of 21 breast cancers. Mutational processes molding the genomes of 21 breast cancers. Roberts, S. Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions.
Cell 46 , — Rausch, T. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations. Cell , 59—71 Stephens, P. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell , 27—40 Korbel, J. Criteria for inference of chromothripsis in cancer genomes. Zhang, C. Chromothripsis from DNA damage in micronuclei. Integrated genomic characterization of papillary thyroid carcinoma. Supek, F. Clustered mutation signatures reveal that error-prone DNA repair targets mutations to active genes.
Mardin, B. A cell-based model system links chromothripsis with hyperploidy. Weischenfeldt, J. Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer. Cancer Cell 23 , — Garsed, D. The architecture and evolution of cancer neochromosomes. Durinck, S. Temporal dissection of tumorigenesis in primary cancers. Cancer Discov. Hayward, N. Whole-genome landscapes of major melanoma subtypes. The Cancer Genome Atlas Network.
Genomic classification of cutaneous melanoma. PubMed Central Google Scholar. Signatures of mutational processes in human cancer. Chan, K. Middlebrooks, C. Westra, H. Systematic identification of trans eQTLs as putative drivers of known disease associations. Stranger, B. Population genomics of human gene expression. Menghi, F. The tandem duplicator phenotype as a distinct genomic configuration in cancer. Natl Acad. USA , E—E Hendrich, B. Lee, E. Landscape of somatic retrotransposition in human cancers. Tubio, J. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.
Helman, E. Somatic retrotransposition in human cancer revealed by whole-genome and exome sequencing. Shay, J. Hayflick, his limit, and cellular ageing. Cell Biol. Peifer, M. Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Totoki, Y. Trans-ancestry mutational landscape of hepatocellular carcinoma genomes. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 22 , — PubMed Google Scholar. Heaphy, C. Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. Barthel, F. Systematic analysis of telomere length and somatic alterations in 31 cancer types. A role for the Rb family of proteins in controlling telomere length.
Tomasetti, C. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science , 78—81 Precision oncology for acute myeloid leukemia using a knowledge bank approach. The Dockstore: enabling modular, community-focused sharing of Docker-based genomics tools and workflows. Zhang, J. Miller, C. Methods 11 , — Goldman, M. Papatheodorou, I. Expression Atlas: gene and protein expression across multiple studies and organisms. Nucleic Acids Res. Li, H. Fast and accurate long-read alignment with Burrows—Wheeler transform.
Bioinformatics 26 , — A global reference for human genetic variation. Nature , 68—74 Raine, K. Bioinformatics 56 , Jones, D. Bioinformatics 52 , Ye, K. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25 , — DELLY: structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics 28 , i—i Rimmer, A. Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Cibulskis, K. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples.
Carter, S. Absolute quantification of somatic DNA alterations in human cancer. Drier, Y. Somatic rearrangements across cancer reveal classes of samples with distinct patterns of DNA breakage and rearrangement-induced hypermutability. Ramos, A. Oncotator: cancer variant annotation tool. Moncunill, V. Comprehensive characterization of complex structural variations in cancer by directly comparing genome sequence reads.
Fan, Y. MuSE: accounting for tumor heterogeneity using a sample-specific error model improves sensitivity and specificity in mutation calling from sequencing data. Genome Biol. Cooke, S. Processed pseudogenes acquired somatically during cancer development. Ju, Y. Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer. Sudmant, P. An integrated map of structural variation in 2, human genomes. Nature , 75—81 Garrison, E. Haplotype-based variant detection from short-read sequencing.
DePristo, M. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Yakneen, S. Butler enables rapid cloud-based analysis of thousands of human genomes. Kim, S. Combining calls from multiple somatic mutation-callers. BMC Bioinformatics 15 , Breiman, L. Stacked regressions. Campbell, P. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Wala, J. SvABA: genome-wide detection of structural variants and indels by local assembly.
Download references. We thank research participants who donated samples and data, the physicians and clinical staff who contributed to sample annotation and collection, and the numerous funding agencies that contributed to the collection and analysis of this dataset. A list of members and their affiliations appears in the online version of the paper and lists of working groups appear in the Supplementary Information. These authors jointly supervised this work: Peter J. Campbell, Gad Getz, Jan O. Korbel, Joshua M. Stuart, Lincoln D. Peter J. Campbell, Keiran M.
Raine, Adam P. Wedge, Maxime Tarabichi, Nicola D. Roberts, Yilong Li, Ludmil B. Alexandrov, Jonathan Hinton, David R. Adams, Kevin J. Dawson, Stefan C. Mitchell, Shriram G. Maruvka, Chip Stewart, Jeremiah A. Wala, Julian M. Hess, Mara Rosenberg, Andrew J. Schumacher, Michael S. Gabriel, Nils Gehlenborg, David I. Meier, Michael S. Jan O. Korbel, Andy Cafferkey, Steven J. Lincoln D.
Stein, Constance H. Li, Paul C. Boutros, L. Jonathan Dursi, Jared T. Simpson, Solomon I. Wright, Shadrielle M. Espiritu, Christopher M. JacksonWhich of the following sentences is correct and why? One point on the line is chosen as the origin. Read Romans Most Traditionalists read this passage as referring to all humanity, with the idol worship used as a metaphor rather than a specific event. Write your answers in boxesWe read every single response to our post-ticket surveys, and I wanted to address some of the concerns you've raised. A successful business man does nothing to increase his popularity by being prudent with his money. Get answers in as little as 15 minutes. This choice amounts to specifying which side of the origin will be the positive half of the line; the other side is then the negative half.
Animals enrich our lives in so many different ways. Complete passage below with the correct form of the verbs given in the box: Simple Past or Past Read the text, choose a suitable sentence for each blank, and write the letter on the line. As said athwart ships is easy I use the bridge deck at the entrance hatch. Second, we are sure that once you new people begin reading it, you'll go out and get a physical copy.
It is labeled 0. Our team of highly qualified tutors make learning fun and easy and help you toNelson Mandela Questions and Answers Question: When was Nelson Mandela born? Answer: According to his biography at Nobelprize. Hank the remaining line there, Maybe a bungee to hold the hank out of the way. Read breaking headlines covering politics, economics, pop culture, and more.
To make the imperative, use the infinitive of the verb without "to". Making statements based on opinion; back them up with references or personal experience. Look for a common theme or idea in the first lines. Reading in a whole new way reading practice test has 14 questions belongs to the Technology subject. I think this is a world view a lot of us hold until we are able to see the bigger picture. Watch extra video from the ship, read journal entries, and. The funniest thing about him is the way he likes to grow—. See Activity. Improve your students' reading comprehension with ReadWorks. If you want to make it clear [exactly where the highlighted passage begins or ends,] you can use small square brackets in the text, as I did in this sentence, along with the vertical line How to Read Sheet Music for Beginners: One of the first things that any beginning pianist learns to do, is to read music.
D: Reading Passage 1. The story as described today:. Whate'er thy thoughts, or thy heart's workings be,Thy looks should nothing thence, but sweetness tell. Ours must not be the generation that falters. Listen for a mix of answers, indicating confusion. The song was released in onA comprehensive database of more than reading quizzes online, test your knowledge with reading quiz questions.
Line clip also does not line up straight. He was trapped. You can also change it using the keyboard alone. Another way to find answers to your Bible questions. The frontier is an exciting, demanding — and frequently lawless — place to be. In verse 17, the plumb line is defined as justice and righteousness. Here is a worksheet to help students master poetic devices. After you have selected your answer, fill in the corresponding bubble on your answer sheet.
Tali and Mordin fight reaper - death. The unique organization of the book placed this reflection before the actual events, so that it serves to foreshadow what will come. Knowledge is like a garden: If it is not cultivated, it cannot be harvested. Im not looking for someone to give me the answer. Once drained remove the bowl nut ,you can have someone hold the bowl in place while you check the fuel passages. I like to use the right margin and to make my line a right square bracket: ]. Eventually I understood this would only lead us to more trouble. He prefers Monopoly because it requires luck and skill. That offer is automatically generated by this website, not by me. This passage holds the key to understanding the entire novel.
How should you answer the interview question "Tell me about a problem you solved at work. The Spartans held Athens and Thebes, establishing there an oligarchy: nevertheless they lost them. Think carefully before you select an answer. Feel the feelings: Don't dwell on them but also don't wall them off. You should spend about 20 minutes on Questions which are based on Reading Passage below: Why some women cross the finish line ahead of men.
Planting a tree is always a great work for the mankind. It was a tough one. Basic Collocations — Pop these fundamental words into the sentences. Identify the character for each. Multiply at the speed of lightning! This worksheet is full of multiplication problems that your child should try to solve in one minute. He works on the assembly line assembling cars. Herbert Davis, 14 vols. You should always use custom on hold messages, not trite and irritating ones. He is a decorated combat veteran. The story is told in alternating sections of past and present. Yours might be one of them. Juan also likes to play less demanding board games that are based mostly on luck. Students orally read passages designed for one-minute readings several times with appropriate expression and smoothness to increase reading rate, resulting in improved focus on comprehension.
Had you been as wise as bold, Young in limbs, in judgment old, Your answer had not been inscroll'd: Fare you well; your suit is cold. It was this African name that was later on supplemented with the English first name Nelson, given to him by his teacher, Miss Mdingane, as the name he should answer Answer: You need to remove the valve to get rid of all the carbon build-up, replace the gasket and check the mounting area for cracks.
Example answer: Your company stood out when I was researching the leading electronics companies in the country. March 17, by Toronto Star. In Congress, July 4, Check out this study guide for all the common topics, examiner questions, model answers and top tips from our in-house IELTS expert at Online Teachers Now, the teachers were all lined up on the track and ready to race. July 5, The papers are informal and so should not require too much time. That the global economy is slowing down is an understatement. Phone number or keyword you want to search for. Keep the prompt in mid when you read.
Now answer the following questions by choosing correct options: In the last stanza of the poem there is a repetition of the line; 'And miles to go before I sleep. Read the passages and choose the best answer to each question. Click words you want to remove from the sentence. In this economics activity, students will make a choice and identify the opportunity cost. When you answer the phone, be warm, enthusiastic, and professional. Question: I have a Ford F 4. Just click the pencil at the end of the line. Buy Study Guide. Open 24 Hours.
Gin has never tasted so good. Mathematics A geometric figure formed by a point moving along a fixed direction and the reverse direction. Tell students to continue until they get back to the first card. Answer the questions that follow each passage. By Mike Kuchar. It is to state what evidence will be presented, to make it easier for the jurors to understand what is to follow, and to relate parts of the evidence and testimony to the whole; it is not an occasion for argument.
Addiction Treatment Center. We can find out the point of time " early 18th century " at paragraph F, line 5 with the sentence " Tea was relatively expensive until Britain started direct trade with China in the early 18th century. By bb. You can pass through a special passage leading to the platform for trains on the next line you plan to take. Apostolic succession is the line of bishops stretching back to the apostles. D2 — Data input 2. And if you're considering reading "Passage," don't read the Wikipedia article about it first; that article includes in passing a spoiler for the most significant plot twist in the book, about three-quarters of the way through the tale.
Lana Pollack. The line in the middle of the Bible text means that you enabled the parallel Bible reading mode. Why do you think that is? Is there a common theme in the book to which everyone can relate?Connecticut History. In the Catalase Enzyme Lab Report had a choice of five daily papers - from the Socialist Dziennik Ludowy [People's daily] What Is Greed In A Christmas Carol to the Polish Roman Catholic Union's Dziennik Catalase Enzyme Lab Report [Union daily] Catalase Enzyme Lab Report - all of which supported archimedes of syracuse struggles for What Is Greed In A Christmas Carol working conditions and were part of a broader program of Clinical Nurse Specialist Case Study and educational activities. Manzarek called Pamela "Jim's other half" and Clinical Nurse Specialist Case Study, "I never knew another person who could so complement his bizarreness.