CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes.

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods because of the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ∼40% of reads mapping to repeats, increasing those mapping to single-copy regions by ∼2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ∼10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ∼12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed because of low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely related species (L. orientalis). Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions.

Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype-phenotype association analysis.

A very early diagnosis of AlstrÓ§m syndrome by next generation sequencing.

BACKGROUND: Alström syndrome is a rare recessively inherited disorder caused by variants in the ALMS1 gene. It is characterized by multiple organ dysfunction, including cone-rod retinal dystrophy, dilated cardiomyopathy, hearing loss, obesity, insulin resistance, hyperinsulinemia, type 2 diabetes mellitus and systemic fibrosis. Heterogeneity and age-dependent development of clinical manifestations make it difficult to obtain a clear diagnosis, especially in pediatric patients. CASE PRESENTATION: Here we report the case of a girl with Alström syndrome. Genetic examination was proposed at age 22 months when suspected macular degeneration was the only major finding. Next generation sequencing of a panel of genes linked to eye-related pathologies revealed two compound heterozygous variants in the ALMS1 gene. Frameshift variants c.1196_1202del, p.(Thr399Lysfs*11), rs761292021 and c.11310_11313del, (p.Glu3771Trpfs*18), rs747272625 were detected in exons 5 and 16, respectively. Both variants cause frameshifts and generation of a premature stop-codon that probably leads to mRNA nonsense-mediated decay. Validation and segregation of ALMS1 variants were confirmed by Sanger sequencing. CONCLUSIONS: Genetic testing makes it possible, even in childhood, to increase the number of correct diagnoses of patients who have ambiguous phenotypes caused by rare genetic variants. The development of high-throughput sequencing technologies offers an exceptionally valuable screening tool for clear genetic diagnoses and ensures early multidisciplinary management and treatment of the emerging symptoms.

A new semi-slug of the genus Microparmarion from Brunei, discovered, described and DNA-barcoded on citizen-science 'taxon expeditions' (Gastropoda, Stylommatophora, Ariophantidae).

Background: During citizen-science expeditions to the Ulu Temburong National Park, Brunei, several individuals were collected of a semi-slug species of the genus Microparmarion that, based on morphology and in-the-field DNA-barcoding, was found to be an undescribed species. New information: In this paper, we describe Microparmarionsallehi Wu, Ezzwan & Hamdani, n. sp., after field centre supervisor Md Salleh Abdullah Bat. We provide details on the external and internal reproductive morphology, the shell and the ecology of the type locality, as well as a diagnosis comparing it with related species. DNA barcodes were generated for five individuals and used for a phylogenetic reconstruction. Microparmarionsallehi sp. n. and M.exquadratus Schilthuizen et al., 2019 so far are the only Bornean species of the genus that live in lowland forest; other species are found in montane forests.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

A fast, reliable and easy method to detect within-species DNA contamination.

BACKGROUND AND AIM: Next generation sequencing (ngs) is becoming the standard for clinical diagnosis. Different steps of NGS, such as DNA extraction, fragmentation, library preparation and amplification, require handling of samples, making the process susceptible to contamination. In diagnostic environments, sample contamination with DNA from the same species can lead to errors in diagnosis. Here we propose a simple method to detect within-sample contamination based on analysis of the heterozygous single nucleotide polymorphisms allele ratio (AR). METHODS: A dataset of 38000 heterozygous snps was used to estimate the ar distribution. The parameters of the reference distribution were then used to estimate the contamination probability of a sample. Validation was performed using 12 samples contaminated to different levels. RESULTS: Results show that the method easily detects contamination of 20% or more. The method has a limit of detection of about 10%, threshold below which the number of false positives increases significantly. CONCLUSIONS: The method can be applied to any type of ngs analysis and is useful for quality control. Being fast and easy to implement makes it ideal for inclusion in NGS pipelines to improve quality control of data and make results more robust.

Study of a supplement and a genetic test for lymphedema management.

Malformations in the lymphatic vasculature, injury, surgery, trauma or toxic damage may lead to swelling of the limbs caused by inefficient lymphatic uptake and flow (lymphedema). Lymphedema can be congenital or acquired. Primary lymphedema is rare and caused by mutations in single genes, secondary lymphedema is more common and caused by a trauma in association with a genetic predisposition. We decided to develop a genetic test that would determine the genetic predisposition to the onset of lymphedema and to predict the course of the disease by analyzing polymorphisms involved in leukotriene B4 (LTB4) synthetic pathway, and variants involved in the onset of secondary lymphedema. There are not many compounds available for the treatment of the negative effects of lymph accumulation, we therefore designed a food supplement based on the hydroxytyrosol, that has anti-oxidant, anti-bacterial and anti-inflammatory activities.

appMAGI: A complete laboratory information management system for clinical diagnostics.

BACKGROUND: The increasing demand for genetic testing for clinical diagnosis and research challenges genetic laboratory capacity to track an increasing number of patient samples through all steps of analysis, from sample collection to report generation. This task is usually performed with the help of a laboratory information management system (LIMS), software that makes it possible to collect, store and retrieve laboratory and sample data. To date there are no open-source options that can manage the entire analytical flow of a genetic laboratory. appMAGI seeks to include all the management aspects of a clinical diagnostic laboratory, making it simpler to process many samples while maintaining the high security and quality standards required in clinical diagnostic practice. METHODS: appMAGI is written in python using Django. It is a web application that does not require local installation, making development, updates and maintenance a much easier task. appMAGI runs on the Ubuntu server and uses SQLite as engine database. RESULTS: In this work we describe an innovative LIMS called appMAGI designed to support all aspects of a clinical diagnostic laboratory. appMAGI can track samples throughout the diagnostic workflow and NGS analysis by virtue of a customizable bioinformatics pipeline. It can handle sample non-compliance, manage laboratory stocks, help generate reports and provide insights into sample data by means of special tools. CONCLUSIONS: appMAGI is a LIMS endowed with all the features required to manage thousands of samples. Allowing efficient management of patient samples from sample collection to diagnostic report generation.

Bacteriophages presence in nature and their role in the natural selection of bacterial populations.

Phages are the obligate parasite of bacteria and have complex interactions with their hosts. Phages can live in, modify, and shape bacterial communities by bringing about changes in their abundance, diversity, physiology, and virulence. In addition, phages mediate lateral gene transfer, modify host metabolism and reallocate bacterially-derived biochemical compounds through cell lysis, thus playing an important role in ecosystem. Phages coexist and coevolve with bacteria and have developed several antidefense mechanisms in response to bacterial defense strategies against them. Phages owe their existence to their bacterial hosts, therefore they bring about alterations in their host genomes by transferring resistance genes and genes encoding toxins in order to improve the fitness of the hosts. Application of phages in biotechnology, environment, agriculture and medicines demands a deep insight into the myriad of phage-bacteria interactions. However, to understand their complex interactions, we need to know how unique phages are to their bacterial hosts and how they exert a selective pressure on the microbial communities in nature. Consequently, the present review focuses on phage biology with respect to natural selection of bacterial populations.

Somatic Variant Analysis Identifies Targets for Tailored Therapies in Patients with Vascular Malformations.

Vascular malformations include various disorders characterized by morphological, structural and/or functional alterations of blood and lymph vessels. Most are sporadic, due to somatic mutations. Here, we report a cohort of patients with sporadic and/or unifocal vascular malformations, in whom we carried out next generation sequencing analysis of a panel of genes associated with vascular malformations. The 115 patients analyzed were from different clinical centres. In 37 patients (32%), we found pathogenic mutations: most of these were gain-of-function mutations in PIK3CA (18%, 21/115) and TEK (13/115, 11%). We also found mutations in GNAQ, CCM2 and PTEN. Identifying pathogenic variants in patients with vascular malformations can help improve management, particularly in cases with activating mutations that cause an increase in cell proliferation. Personalized pharmacological treatment, if possible, is now considered preferable to surgery and can help prevent recurrences, i.e., long-term complications of residual malformation or regrowth of tumors. For instance, rapamycin is currently being investigated for the treatment of various vascular malformations associated with hyperactivation of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway.

Syndromic infertility.

Infertility due to genetic mutations that cause other defects, besides infertility, is defined as syndromic. Here we describe three of these disorders for which we perform genetic tests. 1) Hypopituitarism is an endocrine syndrome characterized by reduced or absent secretion of one or more anterior pituitary hormones with consequent dysfunction of the corresponding peripheral glands. Deficiencies in all the hormones is defined as pan-hypopituitarism, lack of two or more hormones is called partial hypopituitarism, whereas absence of a single hormone is defined as selective hypopituitarism. Pan-hypopituitarism is the rarest condition, whereas the other two are more frequent. Several forms exist: congenital, acquired, organic and functional. 2) The correct functioning of the hypothalamic-pituitary-gonadal axis is fundamental for sexual differentiation and development during fetal life and puberty and for normal gonad function. Alteration of the hypothalamic-pituitary system can determine a condition called hypogonadotropic hypogonadism, characterized by normal/low serum levels of the hormones FSH and LH. 3) Primary ciliary dyskinesia is frequently associated with infertility in males because it impairs sperm motility (asthenozoospermia). Primary ciliary dyskinesia is a group of genetically and phenotypically heterogeneous disorders that show morpho-structural alterations of the cilia. Adult women with primary ciliary dyskinesia can be subfertile and have an increased probability of extra-uterine pregnancies. This is due to delayed transport of the oocyte through the uterine tubes.