Postglacial dispersal of Phlebotomus perniciosus into France

Phlebotomus perniciosus was identified morphologically in samples from France and northeast Spain, and individuals were then characterized at three polymorphic isoenzyme loci (by isoelectrofocusing) and at the mitochondrial DNA locus (by comparative DNA sequence analysis of a fragment of the Cytochrome b gene). The four polymorphic loci gave conflicting patterns of population relationships, which can be explained by hypothesizing different amounts of gene introgression at each locus when two distinctive lineages met in southern France or northeast Spain after isolation in southern Italy and Spain during the Pleistocene Ice Ages. P. perniciosus is an important vector of leishmania infantum and so these population differentiation studies are relevant for predicting the emergence and spread of leishmaniasis in relation to environmental changes, including climate

The Quality Sequencing Minimum (QSM): providing comprehensive, consistent, transparent next generation sequencing data quality assurance.

Next generation sequencing (NGS) is routinely used in clinical genetic testing. Quality management of NGS testing is essential to ensure performance is consistently and rigorously evaluated. Three primary metrics are used in NGS quality evaluation: depth of coverage, base quality and mapping quality. To provide consistency and transparency in the utilisation of these metrics we present the Quality Sequencing Minimum (QSM). The QSM defines the minimum quality requirement a laboratory has selected for depth of coverage (C), base quality (B) and mapping quality (M) and can be applied per base, exon, gene or other genomic region, as appropriate. The QSM format is CX_BY(P Y)_MZ(P Z). X is the parameter threshold for C, Y the parameter threshold for B, P Y the percentage of reads that must reach Y, Z the parameter threshold for M, P Z the percentage of reads that must reach Z. The data underlying the QSM is in the BAM file, so a QSM can be easily and automatically calculated in any NGS pipeline. We used the QSM to optimise cancer predisposition gene testing using the TruSight Cancer Panel (TSCP). We set the QSM as C50_B10(85)_M20(95). Test regions falling below the QSM were automatically flagged for review, with 100/1471 test regions QSM-flagged in multiple individuals. Supplementing these regions with 132 additional probes improved performance in 85/100. We also used the QSM to optimise testing of genes with pseudogenes such as PTEN and PMS2. In TSCP data from 960 individuals the median number of regions that passed QSM per sample was 1429 (97%).  Importantly, the QSM can be used at an individual report level to provide succinct, comprehensive quality assurance information about individual test performance. We believe many laboratories would find the QSM useful. Furthermore, widespread adoption of the QSM would facilitate consistent, transparent reporting of genetic test performance by different laboratories

CoverView: a sequence quality evaluation tool for next generation sequencing data.

Quality assurance and quality control are essential for robust next generation sequencing (NGS). Here we present CoverView, a fast, flexible, user-friendly quality evaluation tool for NGS data. CoverView processes mapped sequencing reads and user-specified regions to report depth of coverage, base and mapping quality metrics with increasing levels of detail from a chromosome-level summary to per-base profiles. CoverView can flag regions that do not fulfil user-specified quality requirements, allowing suboptimal data to be systematically and automatically presented for review. It also provides an interactive graphical user interface (GUI) that can be opened in a web browser and allows intuitive exploration of results. We have integrated CoverView into our accredited clinical cancer predisposition gene testing laboratory that uses the TruSight Cancer Panel (TSCP). CoverView has been invaluable for optimisation and quality control of our testing pipeline, providing transparent, consistent quality metric information and automatic flagging of regions that fall below quality thresholds. We demonstrate this utility with TSCP data from the Genome in a Bottle reference sample, which CoverView analysed in 13 seconds. CoverView uses data routinely generated by NGS pipelines, reads standard input formats, and rapidly creates easy-to-parse output text (.txt) files that are customised by a simple configuration file. CoverView can therefore be easily integrated into any NGS pipeline. CoverView and detailed documentation for its use are freely available at github.com/RahmanTeamDevelopment/CoverView/releases and www.icr.ac.uk/CoverView

The ICR96 exon CNV validation series: a resource for orthogonal assessment of exon CNV calling in NGS data.

Detection of deletions and duplications of whole exons (exon CNVs) is a key requirement of genetic testing. Accurate detection of this variant type has proved very challenging in targeted next-generation sequencing (NGS) data, particularly if only a single exon is involved. Many different NGS exon CNV calling methods have been developed over the last five years. Such methods are usually evaluated using simulated and/or in-house data due to a lack of publicly-available datasets with orthogonally generated results. This hinders tool comparisons, transparency and reproducibility. To provide a community resource for assessment of exon CNV calling methods in targeted NGS data, we here present the ICR96 exon CNV validation series. The dataset includes high-quality sequencing data from a targeted NGS assay (the TruSight Cancer Panel) together with Multiplex Ligation-dependent Probe Amplification (MLPA) results for 96 independent samples. 66 samples contain at least one validated exon CNV and 30 samples have validated negative results for exon CNVs in 26 genes. The dataset includes 46 exon CNVs in BRCA1, BRCA2, TP53, MLH1, MSH2, MSH6, PMS2, EPCAM or PTEN, giving excellent representation of the cancer predisposition genes most frequently tested in clinical practice. Moreover, the validated exon CNVs include 25 single exon CNVs, the most difficult type of exon CNV to detect. The FASTQ files for the ICR96 exon CNV validation series can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428

CSN and CAVA: variant annotation tools for rapid, robust next-generation sequencing analysis in the clinical setting.

Next-generation sequencing (NGS) offers unprecedented opportunities to expand clinical genomics. It also presents challenges with respect to integration with data from other sequencing methods and historical data. Provision of consistent, clinically applicable variant annotation of NGS data has proved difficult, particularly of indels, an important variant class in clinical genomics. Annotation in relation to a reference genome sequence, the DNA strand of coding transcripts and potential alternative variant representations has not been well addressed. Here we present tools that address these challenges to provide rapid, standardized, clinically appropriate annotation of NGS data in line with existing clinical standards.We developed a clinical sequencing nomenclature (CSN), a fixed variant annotation consistent with the principles of the Human Genome Variation Society (HGVS) guidelines, optimized for automated variant annotation of NGS data. To deliver high-throughput CSN annotation we created CAVA (Clinical Annotation of VAriants), a fast, lightweight tool designed for easy incorporation into NGS pipelines. CAVA allows transcript specification, appropriately accommodates the strand of a gene transcript and flags variants with alternative annotations to facilitate clinical interpretation and comparison with other datasets. We evaluated CAVA in exome data and a clinical BRCA1/BRCA2 gene testing pipeline.CAVA generated CSN calls for 10,313,034 variants in the ExAC database in 13.44 hours, and annotated the ICR1000 exome series in 6.5 hours. Evaluation of 731 different indels from a single individual revealed 92 % had alternative representations in left aligned and right aligned data. Annotation of left aligned data, as performed by many annotation tools, would thus give clinically discrepant annotation for the 339 (46 %) indels in genes transcribed from the forward DNA strand. By contrast, CAVA provides the correct clinical annotation for all indels. CAVA also flagged the 370 indels with alternative representations of a different functional class, which may profoundly influence clinical interpretation. CAVA annotation of 50 BRCA1/BRCA2 gene mutations from a clinical pipeline gave 100 % concordance with Sanger data; only 8/25 BRCA2 mutations were correctly clinically annotated by other tools.CAVA is a freely available tool that provides rapid, robust, high-throughput clinical annotation of NGS data, using a standardized clinical sequencing nomenclature

Implementing rapid, robust, cost-effective, patient-centred, routine genetic testing in ovarian cancer patients.

Advances in DNA sequencing have made genetic testing fast and affordable, but limitations of testing processes are impeding realisation of patient benefits. Ovarian cancer exemplifies the potential value of genetic testing and the shortcomings of current pathways to access testing. Approximately 15% of ovarian cancer patients have a germline BRCA1 or BRCA2 mutation which has substantial implications for their personal management and that of their relatives. Unfortunately, in most countries, routine implementation of BRCA testing for ovarian cancer patients has been inconsistent and largely unsuccessful. We developed a rapid, robust, mainstream genetic testing pathway in which testing is undertaken by the trained cancer team with cascade testing to relatives performed by the genetics team. 207 women with ovarian cancer were offered testing through the mainstream pathway. All accepted. 33 (16%) had a BRCA mutation. The result informed management of 79% (121/154) women with active disease. Patient and clinician feedback was very positive. The pathway offers a 4-fold reduction in time and 13-fold reduction in resource requirement compared to the conventional testing pathway. The mainstream genetic testing pathway we present is effective, efficient and patient-centred. It can deliver rapid, robust, large-scale, cost-effective genetic testing of BRCA1 and BRCA2 and may serve as an exemplar for other genes and other diseases

Sarcoma and the 100,000 Genomes Project: our experience and changes to practice

The largest whole genome sequencing (WGS) endeavour involving cancer and rare diseases was initiated in the UK in 2015 and ran for 5 years. Despite its rarity, sarcoma ranked third overall among the number of patients' samples sent for sequencing. Herein, we recount the lessons learned by a specialist sarcoma centre that recruited close to 1000 patients to the project, so that we and others may learn from our experience. WGS data was generated from 597 patients, but samples from the remaining approximately 400 patients were not sequenced. This was largely accounted for by unsuitability due to extensive necrosis, secondary to neoadjuvant radiotherapy or chemotherapy, or being placed in formalin. The number of informative genomes produced was reduced further by a PCR amplification step. We showed that this loss of genomic data could be mitigated by sequencing whole genomes from needle core biopsies. Storage of resection specimens at 4 °C for up to 96 h overcame the challenge of freezing tissue out of hours including weekends. Removing access to formalin increased compliance to these storage arrangements. With over 70 different sarcoma subtypes described, WGS was a useful tool for refining diagnoses and identifying novel alterations. Genomes from 350 of the cohort of 597 patients were analysed in this study. Overall, diagnoses were modified for 3% of patients following review of the WGS findings. Continued refinement of the variant-calling bioinformatic pipelines is required as not all alterations were identified when validated against histology and standard of care diagnostic tests. Further research is necessary to evaluate the impact of germline mutations in patients with sarcoma, and sarcomas with evidence of hypermutation. Despite 50% of the WGS exhibiting domain 1 alterations, the number of patients with sarcoma who were eligible for clinical trials remains small, highlighting the need to revaluate clinical trial design

Identification of new Wilms tumour predisposition genes: an exome sequencing study

BACKGROUND: Wilms tumour is the most common childhood renal cancer and is genetically heterogeneous. While several Wilms tumour predisposition genes have been identified, there is strong evidence that further predisposition genes are likely to exist. Our study aim was to identify new predisposition genes for Wilms tumour. METHODS: In this exome sequencing study, we analysed lymphocyte DNA from 890 individuals with Wilms tumour, including 91 affected individuals from 49 familial Wilms tumour pedigrees. We used the protein-truncating variant prioritisation method to prioritise potential disease-associated genes for further assessment. We evaluated new predisposition genes in exome sequencing data that we generated in 334 individuals with 27 other childhood cancers and in exome data from The Cancer Genome Atlas obtained from 7632 individuals with 28 adult cancers. FINDINGS: We identified constitutional cancer-predisposing mutations in 33 individuals with childhood cancer. The three identified genes with the strongest signal in the protein-truncating variant prioritisation analyses were TRIM28, FBXW7, and NYNRIN. 21 of 33 individuals had a mutation in TRIM28; there was a strong parent-of-origin effect, with all ten inherited mutations being maternally transmitted (p=0·00098). We also found a strong association with the rare epithelial subtype of Wilms tumour, with 14 of 16 tumours being epithelial or epithelial predominant. There were no TRIM28 mutations in individuals with other childhood or adult cancers. We identified truncating FBXW7 mutations in four individuals with Wilms tumour and a de-novo non-synonymous FBXW7 mutation in a child with a rhabdoid tumour. Biallelic truncating mutations in NYNRIN were identified in three individuals with Wilms tumour, which is highly unlikely to have occurred by chance (p<0·0001). Finally, we identified two de-novo KDM3B mutations, supporting the role of KDM3B as a childhood cancer predisposition gene. INTERPRETATION: The four new Wilms tumour predisposition genes identified-TRIM28, FBXW7, NYNRIN, and KDM3B-are involved in diverse biological processes and, together with the other 17 known Wilms tumour predisposition genes, account for about 10% of Wilms tumour cases. The overlap between these 21 constitutionally mutated predisposition genes and 20 genes somatically mutated in Wilms tumour is limited, consisting of only four genes. We recommend that all individuals with Wilms tumour should be offered genetic testing and particularly, those with epithelial Wilms tumour should be offered TRIM28 genetic testing. Only a third of the familial Wilms tumour clusters we analysed were attributable to known genes, indicating that further Wilms tumour predisposition factors await discovery. FUNDING: Wellcome Trust

Explorative visual analytics on interval-based genomic data and their metadata

Background: With the wide-spreading of public repositories of NGS processed data, the availability of user-friendly and effective tools for data exploration, analysis and visualization is becoming very relevant. These tools enable interactive analytics, an exploratory approach for the seamless "sense-making" of data through on-the-fly integration of analysis and visualization phases, suggested not only for evaluating processing results, but also for designing and adapting NGS data analysis pipelines. Results: This paper presents abstractions for supporting the early analysis of NGS processed data and their implementation in an associated tool, named GenoMetric Space Explorer (GeMSE). This tool serves the needs of the GenoMetric Query Language, an innovative cloud-based system for computing complex queries over heterogeneous processed data. It can also be used starting from any text files in standard BED, BroadPeak, NarrowPeak, GTF, or general tab-delimited format, containing numerical features of genomic regions; metadata can be provided as text files in tab-delimited attribute-value format. GeMSE allows interactive analytics, consisting of on-the-fly cycling among steps of data exploration, analysis and visualization that help biologists and bioinformaticians in making sense of heterogeneous genomic datasets. By means of an explorative interaction support, users can trace past activities and quickly recover their results, seamlessly going backward and forward in the analysis steps and comparative visualizations of heatmaps. Conclusions: GeMSE effective application and practical usefulness is demonstrated through significant use cases of biological interest. GeMSE is available at http://www.bioinformatics.deib.polimi.it/GeMSE/ , and its source code is available at https://github.com/Genometric/GeMSEunder GPLv3 open-source license

The Tatton-Brown-Rahman Syndrome: A clinical study of 55 individuals with de novo constitutive DNMT3A variants.

Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous DNMT3A variants. Here we have undertaken a detailed clinical study of 55 individuals with de novoDNMT3A variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS