A Novel Rare Missense Variation of the NOD2 Gene: Evidencesof Implication in Crohn's Disease.

The NOD2 gene, involved in innate immune responses to bacterial peptidoglycan, has been found to be closely associated with Crohn's Disease (CD), with an Odds Ratio ranging from 3⁻36. Families with three or more CD-affected members were related to a high frequency of NOD2 gene variations, such as R702W, G908R, and 1007fs, and were reported in the EPIMAD Registry. However, some rare CD multiplex families were described without identification of common NOD2 linked-to-disease variations. In order to identify new genetic variation(s) closely linked with CD, whole exome sequencing was performed on available subjects, comprising four patients in two generations affected with Crohn's disease without R702W and G908R variation and three unaffected related subjects. A rare and, not yet, reported missense variation of the NOD2 gene, N1010K, was detected and co-segregated across affected patients. In silico evaluation and modelling highlighted evidence for an adverse effect of the N1010K variation with regard to CD. Moreover, cumulative characterization of N1010K and 1007fs as a compound heterozygous state in two, more severe CD family members strongly suggests that N1010K could well be a new risk factor involved in Crohn's disease genetic susceptibility.

A novel NEUROG3 mutation in neonatal diabetes associated with a neuro-intestinal syndrome.

Neonatal diabetes mellitus (NDM) is a rare form of non-autoimmune diabetes usually diagnosed in the first 6 months of life. Various genetic defects have been shown to cause NDM with diverse clinical presentations and variable severity. Among transcriptional factor genes associated with isolated or syndromic NDM, a few cases of homozygous mutations in the NEUROG3 gene have been reported, all mutated patients presenting with congenital malabsorptive diarrhea with or without diabetes at a variable age of onset from early life to childhood. Through a targeted next-generation sequencing assay for monogenic diabetes genes, we aimed to search for pathogenic deleterious mutation in a Turkish patient with NDM, severe malabsorptive diarrhea, neurointestinal dysplasia and other atypical features. In this patient, we identified a novel homozygous nonsense mutation (p.Q4*) in NEUROG3. The same biallelic mutation was found in another affected family member. Of note, the study proband presents with abnormalities of the intrahepatic biliary tract, thyroid gland and central nervous system, which has never been reported before in NEUROG3 mutation carriers. Our findings extend the usually described clinical features associated with NEUROG3 deficiency in humans, and question the extent to which a complete lack of NEUROG3 expression may affect pancreas endocrine function in humans.

Shared genetic predisposition in rheumatoid arthritis-interstitial lung disease and familial pulmonary fibrosis.

Despite its high prevalence and mortality, little is known about the pathogenesis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Given that familial pulmonary fibrosis (FPF) and RA-ILD frequently share the usual pattern of interstitial pneumonia and common environmental risk factors, we hypothesised that the two diseases might share additional risk factors, including FPF-linked genes. Our aim was to identify coding mutations of FPF-risk genes associated with RA-ILD.We used whole exome sequencing (WES), followed by restricted analysis of a discrete number of FPF-linked genes and performed a burden test to assess the excess number of mutations in RA-ILD patients compared to controls.Among the 101 RA-ILD patients included, 12 (11.9%) had 13 WES-identified heterozygous mutations in the TERT, RTEL1, PARN or SFTPC coding regions. The burden test, based on 81 RA-ILD patients and 1010 controls of European ancestry, revealed an excess of TERT, RTEL1, PARN or SFTPC mutations in RA-ILD patients (OR 3.17, 95% CI 1.53-6.12; p=9.45×10-4). Telomeres were shorter in RA-ILD patients with a TERT, RTEL1 or PARN mutation than in controls (p=2.87×10-2).Our results support the contribution of FPF-linked genes to RA-ILD susceptibility.

RSAT 2015: Regulatory Sequence Analysis Tools.

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/.

TMEM240 mutations cause spinocerebellar ataxia 21 with mental retardation and severe cognitive impairment.

Autosomal dominant cerebellar ataxia corresponds to a clinically and genetically heterogeneous group of neurodegenerative disorders that primarily affect the cerebellum. Here, we report the identification of the causative gene in spinocerebellar ataxia 21, an autosomal-dominant disorder previously mapped to chromosome 7p21.3-p15.1. This ataxia was firstly characterized in a large French family with slowly progressive cerebellar ataxia, accompanied by severe cognitive impairment and mental retardation in two young children. Following the recruitment of 12 additional young family members, linkage analysis enabled us to definitively map the disease locus to chromosome 1p36.33-p36.32. The causative mutation, (c.509C>T/p.P170L) in the transmembrane protein gene TMEM240, was identified by whole exome sequencing and then was confirmed by Sanger sequencing and co-segregation analyses. Index cases from 368 French families with autosomal-dominant cerebellar ataxia were also screened for mutations. In seven cases, we identified a range of missense mutations (c.509C>T/p.P170L, c.239C>T/p.T80M, c.346C>T/p.R116C, c.445G>A/p.E149K, c.511C>T/p.R171W), and a stop mutation (c.489C>G/p.Y163*) in the same gene. TMEM240 is a small, strongly conserved transmembrane protein of unknown function present in cerebellum and brain. Spinocerebellar ataxia 21 may be a particular early-onset disease associated with severe cognitive impairment.

RSAT peak-motifs: motif analysis in full-size ChIP-seq datasets.

ChIP-seq is increasingly used to characterize transcription factor binding and chromatin marks at a genomic scale. Various tools are now available to extract binding motifs from peak data sets. However, most approaches are only available as command-line programs, or via a website but with size restrictions. We present peak-motifs, a computational pipeline that discovers motifs in peak sequences, compares them with databases, exports putative binding sites for visualization in the UCSC genome browser and generates an extensive report suited for both naive and expert users. It relies on time- and memory-efficient algorithms enabling the treatment of several thousand peaks within minutes. Regarding time efficiency, peak-motifs outperforms all comparable tools by several orders of magnitude. We demonstrate its accuracy by analyzing data sets ranging from 4000 to 1,28,000 peaks for 12 embryonic stem cell-specific transcription factors. In all cases, the program finds the expected motifs and returns additional motifs potentially bound by cofactors. We further apply peak-motifs to discover tissue-specific motifs in peak collections for the p300 transcriptional co-activator. To our knowledge, peak-motifs is the only tool that performs a complete motif analysis and offers a user-friendly web interface without any restriction on sequence size or number of peaks.

RSAT 2011: regulatory sequence analysis tools.

RSAT (Regulatory Sequence Analysis Tools) comprises a wide collection of modular tools for the detection of cis-regulatory elements in genome sequences. Thirteen new programs have been added to the 30 described in the 2008 NAR Web Software Issue, including an automated sequence retrieval from EnsEMBL (retrieve-ensembl-seq), two novel motif discovery algorithms (oligo-diff and info-gibbs), a 100-times faster version of matrix-scan enabling the scanning of genome-scale sequence sets, and a series of facilities for random model generation and statistical evaluation (random-genome-fragments, random-motifs, random-sites, implant-sites, sequence-probability, permute-matrix). Our most recent work also focused on motif comparison (compare-matrices) and evaluation of motif quality (matrix-quality) by combining theoretical and empirical measures to assess the predictive capability of position-specific scoring matrices. To process large collections of peak sequences obtained from ChIP-seq or related technologies, RSAT provides a new program (peak-motifs) that combines several efficient motif discovery algorithms to predict transcription factor binding motifs, match them against motif databases and predict their binding sites. Availability (web site, stand-alone programs and SOAP/WSDL (Simple Object Access Protocol/Web Services Description Language) web services): http://rsat.ulb.ac.be/rsat/.

Making Bioinformatics Training Events and Material More Discoverable Using TeSS, the ELIXIR Training Portal.

Many trainers and organizations are passionate about sharing their training material. Sharing training material has several benefits, such as providing a record of recognition as an author, offering inspiration to other trainers, enabling researchers to discover training resources for their personal learning path, and improving the training resource landscape using data-driven gap analysis from the bioinformatics community. In this article, we present a series of protocols for using the ELIXIR online training registry Training eSupport System (TeSS). TeSS provides a one-stop shop for trainers and trainees to discover online information and content, including training materials, events, and interactive tutorials. For trainees, we provide protocols for registering and logging in and for searching and filtering content. For trainers and organizations, we also show how to manually or automatically register training events and materials. Following these protocols will contribute to promoting training events and add to a growing catalog of materials. This will concomitantly increase the FAIRness of training materials and events. Training registries like TeSS use a scraping mechanism to aggregate training resources from many providers when they have been annotated using Bioschemas specifications. Finally, we describe how to enrich training resources to allow for more efficient sharing of the structured metadata, such as prerequisites, target audience, and learning outcomes using Bioschemas specification. As increasing training events and material are aggregated in TeSS, searching the registry for specific events and materials becomes crucial. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Searching for training events and materials in TeSS Support Protocol: Integrating TeSS widgets on your website Basic Protocol 2: Logging in to TeSS using an institutional account Alternate Protocol: Creating and logging in to a TeSS account Basic Protocol 3: Manual registration of training events in TeSS Basic Protocol 4: Manual registration of training materials in TeSS Basic Protocol 5: Registration of a content provider in TeSS Basic Protocol 6: Automated harvesting of training events and materials in TeSS.

The EMBRACE web service collection.

The EMBRACE (European Model for Bioinformatics Research and Community Education) web service collection is the culmination of a 5-year project that set out to investigate issues involved in developing and deploying web services for use in the life sciences. The project concluded that in order for web services to achieve widespread adoption, standards must be defined for the choice of web service technology, for semantically annotating both service function and the data exchanged, and a mechanism for discovering services must be provided. Building on this, the project developed: EDAM, an ontology for describing life science web services; BioXSD, a schema for exchanging data between services; and a centralized registry (http://www.embraceregistry.net) that collects together around 1000 services developed by the consortium partners. This article presents the current status of the collection and its associated recommendations and standards definitions.

Retrieve-ensembl-seq: user-friendly and large-scale retrieval of single or multi-genome sequences from Ensembl.

UNLABELLED: The preparation of an appropriate sequence dataset is the starting point of all genomic analyses. We present retrieve-ensembl-seq, an application that considerably eases the retrieval of sequences from the Ensembl database, via our user-friendly web site or web services. The user provides Ensembl identifiers or gene names, and the program returns corresponding upstream, downstream, intronic, exonic, UTR or whole gene sequences. retrieve-ensembl-seq also offers a multiple organism mode to retrieve sequences from homologous genes at any taxonomical level. And we introduce various original filters such as the masking of coding fragments and the avoidance of sequence redundancy for genes with multiple transcripts. retrieve-ensembl-seq is included in the software suite regulatory sequence analysis tools (RSAT), allowing instant submission of retrieved sequences to further analysis tools. AVAILABILITY: retrieve-ensembl-seq is integrated in the RSAT suite: http://rsat.ulb.ac.be/rsat. Web site: http://rsat.ulb.ac.be/rsat/retrieve-ensembl-seq_form.cgi. Web services: http://rsat.ulb.ac.be/rsat/web_services/RSATWS.wsdl. Stand-alone distribution: freely available under an academic licence to download from the RSAT web site. The complete manual, a convenient tutorial and demos are available from the RSAT website. Additional help can be found on the RSAT public forum.

NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.

The network analysis tools (NeAT) (http://rsat.ulb.ac.be/neat/) provide a user-friendly web access to a collection of modular tools for the analysis of networks (graphs) and clusters (e.g. microarray clusters, functional classes, etc.). A first set of tools supports basic operations on graphs (comparison between two graphs, neighborhood of a set of input nodes, path finding and graph randomization). Another set of programs makes the connection between networks and clusters (graph-based clustering, cliques discovery and mapping of clusters onto a network). The toolbox also includes programs for detecting significant intersections between clusters/classes (e.g. clusters of co-expression versus functional classes of genes). NeAT are designed to cope with large datasets and provide a flexible toolbox for analyzing biological networks stored in various databases (protein interactions, regulation and metabolism) or obtained from high-throughput experiments (two-hybrid, mass-spectrometry and microarrays). The web interface interconnects the programs in predefined analysis flows, enabling to address a series of questions about networks of interest. Each tool can also be used separately by entering custom data for a specific analysis. NeAT can also be used as web services (SOAP/WSDL interface), in order to design programmatic workflows and integrate them with other available resources.

RSAT: regulatory sequence analysis tools.

The regulatory sequence analysis tools (RSAT, http://rsat.ulb.ac.be/rsat/) is a software suite that integrates a wide collection of modular tools for the detection of cis-regulatory elements in genome sequences. The suite includes programs for sequence retrieval, pattern discovery, phylogenetic footprint detection, pattern matching, genome scanning and feature map drawing. Random controls can be performed with random gene selections or by generating random sequences according to a variety of background models (Bernoulli, Markov). Beyond the original word-based pattern-discovery tools (oligo-analysis and dyad-analysis), we recently added a battery of tools for matrix-based detection of cis-acting elements, with some original features (adaptive background models, Markov-chain estimation of P-values) that do not exist in other matrix-based scanning tools. The web server offers an intuitive interface, where each program can be accessed either separately or connected to the other tools. In addition, the tools are now available as web services, enabling their integration in programmatic workflows. Genomes are regularly updated from various genome repositories (NCBI and EnsEMBL) and 682 organisms are currently supported. Since 1998, the tools have been used by several hundreds of researchers from all over the world. Several predictions made with RSAT were validated experimentally and published.

High Prevalence of Rare Monogenic Forms of Obesity in Obese Guadeloupean Afro-Caribbean Children.

Context: The population of Guadeloupe Island exhibits a high prevalence of obesity. Objective: We aimed to investigate whether rare genetic mutations in genes involved in monogenic obesity (or diabetes) might be causal in this population of Afro-Caribbean ancestry. Design and Setting: This was a secondary analysis of a study on obesity conducted in schoolchildren from Guadeloupe in 2013 that aimed to assess changes in children's profiles after a lifestyle intervention program. Through next-generation sequencing, we sequenced coding regions of 59 genes involved in monogenic obesity or diabetes in participants from this study. Participants and Interventions: A total of 25 obese schoolchildren from Guadeloupe were screened for rare mutations (nonsynonymous, splice-site, or insertion/deletion) in 59 genes. Main Outcome Measures: Correlation between phenotypes and mutations of interest. Results: We detected five rare heterozygous mutations in five different children with obesity: MC4R p.Ile301Thr and SIM1 p.Val326Thrfs*43 mutations that were pathogenic; SIM1 p.Ser343Pro and SH2B1 p.Pro90His mutations that were likely pathogenic; and NTRK2 p.Leu140Phe that was of uncertain significance. In parallel, we identified seven carriers of mutations in ABCC8 (p.Lys1521Asn and p.Ala625Val) or KCNJ11 (p.Val13Met and p.Val151Met) that were of uncertain significance. Conclusions: We were able to detect pathogenic or likely pathogenic mutations linked to severe obesity in >15% of this population, which is much higher than what we observed in Europeans (∼5%).

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145). RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

Loss-of-function mutations in ADCY3 cause monogenic severe obesity.

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight 1 . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations 2 . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3-5. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

Increased Hepatic PDGF-AA Signaling Mediates Liver Insulin Resistance in Obesity-Associated Type 2 Diabetes.

In type 2 diabetes (T2D), hepatic insulin resistance is strongly associated with nonalcoholic fatty liver disease (NAFLD). In this study, we hypothesized that the DNA methylome of livers from patients with T2D compared with livers of individuals with normal plasma glucose levels can unveil some mechanism of hepatic insulin resistance that could link to NAFLD. Using DNA methylome and transcriptome analyses of livers from obese individuals, we found that hypomethylation at a CpG site in PDGFA (encoding platelet-derived growth factor α) and PDGFA overexpression are both associated with increased T2D risk, hyperinsulinemia, increased insulin resistance, and increased steatohepatitis risk. Genetic risk score studies and human cell modeling pointed to a causative effect of high insulin levels on PDGFA CpG site hypomethylation, PDGFA overexpression, and increased PDGF-AA secretion from the liver. We found that PDGF-AA secretion further stimulates its own expression through protein kinase C activity and contributes to insulin resistance through decreased expression of insulin receptor substrate 1 and of insulin receptor. Importantly, hepatocyte insulin sensitivity can be restored by PDGF-AA-blocking antibodies, PDGF receptor inhibitors, and by metformin, opening therapeutic avenues. Therefore, in the liver of obese patients with T2D, the increased PDGF-AA signaling contributes to insulin resistance, opening new therapeutic avenues against T2D and possibly NAFLD.

Discovery of motifs in promoters of coregulated genes.

We present a method to predict cis-acting elements by detecting over-represented motifs in promoters of a set of coregulated genes (single-genome, multigenes approach). The method has been used successfully to detect regulating elements in bacteria and yeast. It can be used with higher organisms as well, but with a loss in reliability of the predictions. A web interface is available at the Regulatory Sequence Analysis Tools site (http://rsat.scmbb.ulb.ac.be/rsat/).

Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion.

OBJECTIVES: Genome-wide association studies (GWAS) have identified >100 loci independently contributing to type 2 diabetes (T2D) risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. METHODS: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-ßH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq) so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-ßH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. RESULTS: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-ßH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19) with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6) with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-ßH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the expression of Prc1, Srr, Zfand6, and Zfand3 was found in mouse pancreatic islets with altered beta-cell function. CONCLUSIONS: This study showed the ability of post-GWAS functional studies to identify new genes and pathways involved in human pancreatic beta-cell function and in T2D pathophysiology.

KAT2B Is Required for Pancreatic Beta Cell Adaptation to Metabolic Stress by Controlling the Unfolded Protein Response.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic ß cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and ß cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent ß cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic ß cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive ß cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.