Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation.

The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .

A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations.

BACKGROUND: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS: UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana.

Basic helix-loop-helix (bHLH) transcription factors are involved in a wide range of developmental processes and in response to biotic and abiotic stresses. They represent one of the biggest families of transcription factors but only few of them have been functionally characterized. Here we report the characterization of AtbHLH68 and show that, although the knock out mutant did not have an obvious development phenotype, it was slightly more sensitive to drought stress than the Col-0, and AtbHLH68 overexpressing lines displayed defects in lateral root (LR) formation and a significant increased tolerance to drought stress, likely related to an enhanced sensitivity to abscisic acid (ABA) and/or increased ABA content. AtbHLH68 was expressed in the vascular system of Arabidopsis and its expression was modulated by exogenously applied ABA in an organ-specific manner. We showed that the expression of genes involved in ABA metabolism [AtAAO3 (AtALDEHYDE OXIDASE 3) and AtCYP707A3 (AtABSCISIC ACID 8'HYDROXYLASE 3)], in ABA-related response to drought-stress (AtMYC2, AtbHLH122 and AtRD29A) or during LRs development (AtMYC2 and AtABI3) was de-regulated in the overexpressing lines. We propose that AtbHLH68 has a function in the regulation of LR elongation, and in the response to drought stress, likely through an ABA-dependent pathway by regulating directly or indirectly components of ABA signaling and/or metabolism.

The non-DNA-binding bHLH transcription factor PRE3/bHLH135/ATBS1/TMO7 is involved in the regulation of light signaling pathway in Arabidopsis.

Plant basic Helix-loop-helix (bHLH) proteins are transcription factors that are involved in many developmental mechanisms, including light signaling and hormone homeostasis. Some of them are non-DNA-binding proteins and could act as dominant negative regulators of other bHLH proteins by forming heterodimers, in a similar way to animal inhibitor of DNA-binding proteins. It has been recently reported that several non-DNA-binding bHLHs are involved in light signaling (KDR/PRE6), gibberellic acid signaling (PRE1/BNQ1/bHLH136) or brassinosteroid signaling (ATBS1). Here we report that Arabidopsis lines overexpressing the PRE3/bHLH135/ATBS1/TMO7 gene are less responsive to red, far-red and blue light than wild-type which is likely to explain the light hyposensitive phenotype displayed when grown under white light conditions. Using quantitative polymerase chain reaction, we show that the expression of PRE3 and KDR/PRE6 genes is regulated by light and that light-related genes are deregulated in the PRE3-ox lines. We show that PRE3 is expressed in the shoot and root meristems and that PRE3-ox lines also have a defect in lateral root development. Our results not only suggest that PRE3 is involved in the regulation of light signaling, but also support the hypothesis that non-DNA-binding bHLH genes are promiscuous genes regulating a wide range of both overlapping and specific regulatory pathways.

Combined networks regulating seed maturation.

Seed maturation is an important phase of seed development during which embryo growth ceases, storage products accumulate, the protective tegument differentiates and tolerance to desiccation develops, leading to seed dormancy. The spatial and temporal regulation of all these processes requires the concerted action of several signaling pathways that integrate information from genetic programs, and both hormonal and metabolic signals. Recent genetic studies have identified some of the interactions that occur between four master regulators in Arabidopsis, increasing our knowledge of the control of the transcriptional program involved in seed maturation. Moreover, several recent breakthroughs have led to a better understanding of the role of abscisic acid signal modulation and the importance of metabolic regulation in the maternal to filial switch leading to the maturation phase.

Identification of new gene expression regulators specifically expressed during plant seed maturation.

A cDNA-AFLP approach on Linum usitatissimum (flax) was used to identify genes specifically expressed during the seed maturation process. Among the 20,000 cDNA-AFLP tags produced, 486 were selected for their seed-specific expression during maturation. When compared with the publicly available databases, half of them presented some significant similarity with known plant sequences. The results obtained confirmed the accuracy of the approach as numerous genes previously described as being expressed exclusively in plant seeds were identified in this screen. The focus was on sequences similar to plant regulators involved in the control of gene expression, either at the transcriptional, post-transcriptional, or post-translational levels. Using a real-time RT-PCR approach, seed-specific expression kinetics were confirmed for 13 of these regulators that were never characterized for being expressed during seed maturation. Among these, a flax gene of the non-LEC1-like HAP3 family and a flax MYB factor were shown to be expressed in specialized tissues of flax embryo using an in situ hybridization approach. By expression kinetic comparison between these flax genes and their Arabidopsis counterparts, it was found that the new HAP3 gene should be related to a ubiquitous seed maturation mechanism, while a new MYB factor appears to be related to a more seed-specific maturation mechanism. These results demonstrate the utility of the flax database in not only identifying new genes expressed during seed maturation but also in being able to highlight the distinction between conserved and non-conserved seed maturation mechanisms.