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Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis-a neglected tropical disease (NTD) affecting ~35 million people worldwide. No vaccine is available, and chemotherapy relies on one anthelmintic, praziquantel. This parasite has a complex life history and is known to infect a range of species of intermediate (freshwater snails and fish) and definitive (piscivorous) hosts. Despite this biological complexity and the impact of this biocarcinogenic pathogen, there has been no previous study of molecular variation in this parasite on a genome-wide scale. Here, we conducted the first extensive nuclear genomic exploration of C. sinensis individuals (n = 152) representing five distinct populations from mainland China, and one from Far East Russia, and revealed marked genetic variation within this species between "northern" and "southern" geographical regions. The discovery of this variation indicates the existence of biologically distinct variants within C. sinensis, which may have distinct epidemiology, pathogenicity and/or chemotherapic responsiveness. The detection of high heterozygosity within C. sinensis specimens suggests that this parasite has developed mechanisms to readily adapt to changing environments and/or host species during its life history/evolution. From an applied perspective, the identification of invariable genes could assist in finding new intervention targets in this parasite, given the major clinical relevance of clonorchiasis. From a technical perspective, the genomic-informatic workflow established herein will be readily applicable to a wide range of other parasites that cause NTDs.
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Clonorquiasis , Clonorchis sinensis , Animales , Clonorchis sinensis/genética , Clonorquiasis/diagnóstico , Clonorquiasis/epidemiología , Clonorquiasis/parasitología , Variación Genética , Asia Oriental , China/epidemiologíaRESUMEN
The application of wastewater-based epidemiology (WBE) to support the global response to the COVID-19 pandemic has shown encouraging outcomes. The accurate, sensitive, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in municipal wastewater is critical for WBE. Here, we present a novel approach based on multiplexed amplicon-based sequencing, namely the ATOPlex platform, for detecting SARS-CoV-2. The ATOPlex platform is capable of quantifying SARS-CoV-2 RNA at concentrations that are at least 1 order of magnitude lower than the detection limit of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Robust and accurate phylogenetic placement can be done at viral concentrations 4 times lower than the detection limit of RT-qPCR. We further found that the solid fraction in wastewater harbors a considerable amount of viral RNA, highlighting the need to extract viral RNA from the solid and liquid fractions of wastewater. This study delivers a highly sensitive, phylogenetically informative, and high-throughput analytical workflow that facilitates the application of WBE.
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BACKGROUND: Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. RESULTS: Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. CONCLUSIONS: The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.
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Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Genoma de Planta , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
Clonorchiasis is a neglected tropical disease caused by the Chinese liver fluke, Clonorchis sinensis, and is often associated with a malignant form of bile duct cancer (cholangiocarcinoma). Although some aspects of the epidemiology of clonorchiasis are understood, little is known about the genetics of C. sinensis populations. Here, we conducted a comprehensive genetic exploration of C. sinensis from endemic geographic regions using complete mitochondrial protein gene sets. Genomic DNA samples from C. sinensis individuals (n = 183) collected from cats and dogs in China (provinces of Guangdong, Guangxi, Hunan, Heilongjiang and Jilin) as well as from rats infected with metacercariae from cyprinid fish from the Russian Far East (Primorsky Krai region) were deep sequenced using the BGISEQ-500 platform. Informatic analyses of mitochondrial protein gene data sets revealed marked genetic variation within C. sinensis; significant variation was identified within and among individual worms from distinct geographical locations. No clear affiliation with a particular location or host species was evident, suggesting a high rate of dispersal of the parasite across endemic regions. The present work provides a foundation for future biological, epidemiological and ecological studies using mitochondrial protein gene data sets, which could aid in elucidating associations between particular C. sinensis genotypes/haplotypes and the pathogenesis or severity of clonorchiasis and its complications (including cholangiocarcinoma) in humans.
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Clonorquiasis/parasitología , Clonorchis sinensis/genética , ADN Mitocondrial/genética , Variación Genética , Animales , China/epidemiología , Clonorquiasis/epidemiología , Haploidia , Interacciones Huésped-Parásitos , Humanos , Filogenia , Federación de Rusia/epidemiologíaRESUMEN
This corrects the article DOI: 10.1038/nature21063.
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The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
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Carcinoma Neuroendocrino/genética , Genoma Humano/genética , Genómica , Neoplasias Pancreáticas/genética , Secuencia de Bases , Proteínas de Unión a Calmodulina/genética , Ensamble y Desensamble de Cromatina/genética , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , ADN Glicosilasas/genética , Análisis Mutacional de ADN , Reparación del ADN/genética , Femenino , Mutación de Línea Germinal/genética , Humanos , Masculino , Proteína EWS de Unión a ARN , Proteínas de Unión al ARN/genética , Serina-Treonina Quinasas TOR/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.
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Carcinoma Ductal Pancreático/genética , Reparación de la Incompatibilidad de ADN/genética , Mutación , Neoplasias Pancreáticas/genética , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Genoma , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.
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Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Genómica , Mutación/genética , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Ductal Pancreático/clasificación , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Metilación de ADN , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-gamma del Hepatocito/genética , Histona Demetilasas/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Ratones , Proteínas Nucleares/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Supervivencia , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas de Pez CebraRESUMEN
The ampulla of Vater is a complex cellular environment from which adenocarcinomas arise to form a group of histopathologically heterogenous tumors. To evaluate the molecular features of these tumors, 98 ampullary adenocarcinomas were evaluated and compared to 44 distal bile duct and 18 duodenal adenocarcinomas. Genomic analyses revealed mutations in the WNT signaling pathway among half of the patients and in all three adenocarcinomas irrespective of their origin and histological morphology. These tumors were characterized by a high frequency of inactivating mutations of ELF3, a high rate of microsatellite instability, and common focal deletions and amplifications, suggesting common attributes in the molecular pathogenesis are at play in these tumors. The high frequency of WNT pathway activating mutation, coupled with small-molecule inhibitors of ß-catenin in clinical trials, suggests future treatment decisions for these patients may be guided by genomic analysis.
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Adenocarcinoma/genética , Proteínas de Unión al ADN/genética , Neoplasias Duodenales/genética , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genética , Vía de Señalización Wnt , Adenocarcinoma/metabolismo , Ampolla Hepatopancreática/patología , Secuencia de Bases , Neoplasias Duodenales/metabolismo , Inestabilidad Genómica , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Neoplasias Pancreáticas/metabolismoRESUMEN
Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/enzimología , Análisis Mutacional de ADN , Activación Enzimática , Amplificación de Genes , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Ligandos , Terapia Molecular Dirigida , Mutación , Fenotipo , Fosforilación , Medicina de Precisión , Valor Predictivo de las Pruebas , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , Microambiente TumoralRESUMEN
Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.
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Conotoxinas/genética , Conotoxinas/metabolismo , Caracol Conus/química , Cisteína/metabolismo , Perfilación de la Expresión Génica , Proteómica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Estructuras Animales/metabolismo , Animales , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Codón/genética , Conotoxinas/química , Caracol Conus/anatomía & histología , ADN Complementario/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Familia de Multigenes , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , ARN/genética , ARN/metabolismo , Edición de ARN , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual's phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci.
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ADN/análisis , Perfilación de la Expresión Génica/métodos , ARN/análisis , Análisis de Secuencia de ARN/métodos , Desequilibrio Alélico , Encéfalo/metabolismo , Humanos , Hígado/metabolismo , Polimorfismo de Nucleótido Simple , Isoformas de ARN , Análisis de Secuencia de ADN/métodosRESUMEN
Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.
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Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Genoma Humano/genética , Neoplasias Ováricas/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Estudios de Cohortes , Ciclina E/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Metilación de ADN , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Femenino , Genes BRCA1 , Genes BRCA2 , Genes de Neurofibromatosis 1 , Mutación de Línea Germinal/genética , Humanos , Mutagénesis/genética , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , Regiones Promotoras Genéticas/genética , Proteína de Retinoblastoma/genéticaRESUMEN
Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.
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Análisis Mutacional de ADN , Genoma Humano/genética , Genómica , Mutación/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Reparación del ADN/genética , Femenino , Genes BRCA1 , Genes BRCA2 , Marcadores Genéticos/genética , Inestabilidad Genómica/genética , Genotipo , Humanos , Ratones , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/tratamiento farmacológico , Platino (Metal)/farmacología , Mutación Puntual/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.
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Adenocarcinoma/genética , Adenocarcinoma/patología , Carcinogénesis/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Reordenamiento Génico/genética , Genoma Humano/genética , Carcinogénesis/patología , Rotura Cromosómica , Cromosomas Humanos/genética , Humanos , Mutación/genéticaRESUMEN
Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.
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Puntos de Rotura del Cromosoma , Cromosomas Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencia de Bases , Carcinoma Ductal Pancreático/genética , Aberraciones Cromosómicas , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Neoplasias/genética , Neoplasias Pancreáticas/genética , Reacción en Cadena de la Polimerasa/métodos , Flujo de TrabajoRESUMEN
Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.
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Biología Computacional , Neoplasias/genética , Mutación Puntual/genética , Programas Informáticos , Variaciones en el Número de Copia de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Neoplasias/patología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. METHODS: Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. RESULTS: Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. CONCLUSIONS: ABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma.
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Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.