Nimbus: A design-driven analyses suite for amplicon-based NGS data

Motivation: PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of ampliconbased sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results: We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons

Exome sequencing and functional analyses suggest that SIX6 is a gene involved in an altered proliferation-differentiation balance early in life and optic nerve degeneration at old age

Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10-7, n = 11 473) and POAG (Pmeta = 6.09 × 10-3, n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10-3, n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG

Exome-wide meta-analysis identifies rare 3'-UTR variant in ERCC1/CD3EAP associated with symptoms of sleep apnea

Obstructive sleep apnea (OSA) is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%), there have been only few studies looking into the genetics of OSA. In the present study, we aimed to identify genetic variants associated with symptoms of sleep apnea by performing a whole-exome sequence meta-analysis of symptoms of sleep apnea in 1,475 individuals of European descent. We identified 17 rare genetic variants with at least suggestive evidence of significance. Replication in an independent dataset confirmed the association of a rare genetic variant (rs2229918; minor allele frequency = 0.3%) with symptoms of sleep apnea (p-valuemeta = 6.98 × 10-9, ßmeta = 0.99). Rs2229918 overlaps with the 3' untranslated regions of ERCC1 and CD3EAP genes on chromosome 19q13. Both genes are expressed in tissues in the neck area, such as the tongue, muscles, cartilage and the trachea. Further, CD3EAP is localized in the nucleus and mitochondria and involved in the tumor necrosis factor-alpha/nuclear factor kappa B signaling pathway. Our results and biological functions of CD3EAP/ERCC1 genes suggest that the 19q13 locus is interesting for further OSA research

Deciphering the RNA landscape by RNAome sequencing

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods

Whole-genome linkage scan combined with exome sequencing identifies novel candidate genes for carotid intima-media thickness

Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage analysis of individuals in the extremes of cIMT trait distribution (>90th percentile) in a large family-based study from a genetically isolated population in the Netherlands. Linked regions were subsequently explored by fine-mapping using exome sequencing. We observed significant evidence of linkage on chromosomes 2p16.3 [rs1017418, heterogeneity LOD (HLOD) = 3.35], 19q1343 (rs3499, HLOD = 9.09), 20p13 (rs1434789, HLOD = 4.10), and 21q22.12 (rs2834949, HLOD = 3.59). Fine-mapping using exome sequencing data identified a non-coding variant (rs62165235) in PNPT1 gene under the linkage peak at chromosome 2 that is likely to have a regulatory function. The variant was associated with quantitative cIMT in the family-based study population (effect = 0.27, p-value = 0.013). Furthermore, we identified several genes under the linkage peak at chromosome 21 highly expressed in tissues relevant for atherosclerosis. To conclude, our linkage analysis identified four genomic regions significantly linked to cIMT. Further analyses are needed to demonstrate involvement of identified candidate genes in development of atherosclerosis

Exome Sequencing Analysis Identifies Rare Variants in ATM and RPL8 That Are Associated With Shorter Telomere Length

Telomeres are important for maintaining genomic stability. Telomere length has been associated with aging, disease, and mortality and is highly heritable (∼82%). In this study, we aimed to identify rare genetic variants associated with telomere length using whole-exome sequence data. We studied 1,303 participants of the Erasmus Rucphen Family (ERF) study, 1,259 of the Rotterdam Study (RS), and 674 of the British Heart Foundation Family Heart Study (BHF-FHS). We conducted two analyses, first we analyzed the family-based ERF study and used the RS and BHF-FHS for replication. Second, we combined the summary data of the three studies in a meta-analysis. Telomere length was measured by quantitative polymerase chain reaction in blood. We identified nine rare variants significantly associated with telomere length (p-value < 1.42 × 10–7, minor allele frequency of 0.2–0.5%) in the ERF study. Eight of these variants (in C11orf65, ACAT1, NPAT, ATM, KDELC2, and EXPH5) were located on chromosome 11q22.3 that contains ATM, a gene involved in telomere maintenance. Although we were unable to replicate the variants in the RS and BHF-FHS (p-value ≥ 0.21), segregation analysis showed that all variants segregate with shorter telomere length in a family. In the meta-analysis of all studies, a nominally significant association with LTL was observed with a rare variant in RPL8 (p-value = 1.48 × 10−6), which has previously been associated with age. Additionally, a novel rare variant in the known RTEL1 locus showed suggestive evidence for association (p-value = 1.18 × 10–4) with LTL. To conclude, we identified novel rare variants associated with telomere length. Larger samples size are needed to confirm these findings and to identify additional variants

Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Background: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases

Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping