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PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0250799
Author(s):  
Nicole D. Dueker ◽  
Ashley Beecham ◽  
Liyong Wang ◽  
Chuanhui Dong ◽  
Ralph L. Sacco ◽  
...  

Carotid plaque is a subclinical measure of atherosclerosis. We have previously shown measures of carotid plaque to be heritable in a sample of 100 Dominican families and found evidence for linkage and association of common variants (CVs) on 7q36, 11p15, 14q32 and 15q23 with plaque presence. Our current study aimed to refine these regions further and identify rare variants (RVs) influencing plaque presence. Therefore, we performed targeted sequencing of the one LOD unit down region on 7q36, 11p15, 14q32 and 15q23 in 12 Dominican families with evidence for linkage to plaque presence. Gene-based RV analyses were performed using the Sequence Association Test for familial data (F-SKAT) under two filtering algorithms; 1. all exonic RVs and 2. non-synonymous RVs. Replication analyses were performed using a sample of 22 Dominican families and 556 unrelated Dominicans with Exome Array data. To identify additional non-synonymous RVs influencing plaque, we looked for co-segregation of RVs with plaque in each of the sequenced families. Our most strongly associated gene with evidence for replication was AMPD3 which showed suggestive association with plaque presence in the sequenced families (exonic RV p = 0.003, nonsynonymous RV p = 0.005) and replication families (exonic RV p = 0.04, nonsynonymous RV p = 0.02). Examination of the sequenced family pedigrees revealed two missense variants on chromosome 11 which co-segregated with plaque presence in one of our families; rs61751342 (located in DENND2B), and rs61760882 (located in RNF141). The rs61751342 missense variant is an eQTL for SCUBE2 in the atrial appendage. Notably, SCUBE2 encodes a protein which interacts with vascular endothelial growth factor (VEGF) receptor 2 to regulate VEGF-induced angiogenesis, thus providing biologic plausibility for this gene in atherosclerosis. In conclusion, using targeted sequencing of previously-identified linkage regions, we have identified suggestive evidence for the role of RVs in carotid plaque pathogenesis.


2022 ◽  
Author(s):  
Silas Shumate ◽  
Maggie Haylett ◽  
Brenda Nelson ◽  
Nicole Young ◽  
Kurt Lamour ◽  
...  

Tetranychus urticae (Koch) is an economically important pest of many agricultural commodities in the Pacific Northwest. Multiple miticides are currently registered for control including abamectin, bifenazate, bifenthrin, and extoxazole. However, populations of Tetranychus urticae have developed miticide resistance through multiple mechanisms, in many different growing regions. Producers of agricultural commodities where Tetranychus urticae infestations are problematic rely on integrated pest management tools to determine optimal control methods. Within this species multiple single nucleotide polymorphisms have been documented in different genes which are associated with miticide resistance phenotypes. The detection of these mutations through TaqMan qPCR has been suggested as a practical, quick, and reliable tool to inform agricultural producers of miticide resistance phenotypes present within their fields and have potential utility for making appropriate miticide application and integrated pest management decisions. Within this investigation we examined the use of a TaqMan qPCR-based approach to determine miticide resistance genotypes in field-collected populations of Tetranychus urticae from mint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results suggest the TaqMan approach accurately genotypes Tetranychus urticae populations collected from agricultural fields. The interpretation of the results, however, provide additional challenges for integrated pest management practitioners, including making miticide application recommendations where populations of Tetranychus urticae are a mix of resistant and wildtype individuals.


Author(s):  
Michelle L. Roberts ◽  
Theodore A. Kotchen ◽  
Xiaoqing Pan ◽  
Yingchuan Li ◽  
Chun Yang ◽  
...  

Background: Epigenetic marks (eg, DNA methylation) may capture the effect of gene-environment interactions. DNA methylation is involved in blood pressure (BP) regulation and hypertension development; however, no studies have evaluated its relationship with 24-hour BP phenotypes (daytime, nighttime, and 24-hour average BPs). Methods: We examined the association of whole blood DNA methylation with 24-hour BP phenotypes and clinic BPs in a discovery cohort of 281 Blacks using reduced representation bisulfite sequencing. We developed a deep and region-specific methylation sequencing method, Bisulfite ULtrapLEx Targeted Sequencing and utilized it to validate our findings in a separate validation cohort (n=117). Results: Analysis of 38 215 DNA methylation regions (MRs), derived from 1 549 368 CpG sites across the genome, identified up to 72 regions that were significantly associated with 24-hour BP phenotypes. No MR was significantly associated with clinic BP. Two to 3 MRs were significantly associated with various 24-hour BP phenotypes after adjustment for age, sex, and body mass index. Together, these MRs explained up to 16.5% of the variance of 24-hour average BP, while age, sex, and BMI explained up to 11.0% of the variance. Analysis of one of the MRs in an independent cohort using Bisulfite ULtrapLEx Targeted Sequencing confirmed its association with 24-hour average BP phenotype. Conclusions: We identified several MRs that explain a substantial portion of variances in 24-hour BP phenotypes, which might be excellent markers of cumulative effect of factors influencing 24-hour BP levels. The Bisulfite ULtrapLEx Targeted Sequencing workflow has potential to be suitable for clinical testing and population screenings on a large scale.


Author(s):  
Anna Stadermann ◽  
Martin Gamer ◽  
Jürgen Fieder ◽  
Benjamin Lindner ◽  
Steffen Fehrmann ◽  
...  

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Carla Mariner-Llicer ◽  
Galo A. Goig ◽  
Laura Zaragoza-Infante ◽  
Manuela Torres-Puente ◽  
Luis Villamayor ◽  
...  

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis , identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study’s main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.


Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1979
Author(s):  
Francesco Musacchia ◽  
Marianthi Karali ◽  
Annalaura Torella ◽  
Steve Laurie ◽  
Valeria Policastro ◽  
...  

Homozygous deletions (HDs) may be the cause of rare diseases and cancer, and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking. We present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness, we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms that each missed at least one event. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis. We provide VarGenius-HZD either stand-alone or integrated within our recently developed software, enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes.


2021 ◽  
Vol 7 (6) ◽  
pp. a006121
Author(s):  
Siren Berland ◽  
Jørgen Jareld ◽  
Nicholas Hickson ◽  
Helene Schlecht ◽  
Gunnar Houge ◽  
...  

We report a patient with a germline RIT1 and a mosaic PIK3CA variant. The diagnosis of the RASopathy was confirmed by targeted sequencing following the identification of transient cardiomyopathy in a patient with PIK3CA-related overgrowth spectrum (PROS). Our observation confirms that the PIK3CA gain-of-function (GoF) variant effects dominate those of the RASopathy, and the resulting blended phenotype mostly resembles megalencephaly-capillary malformation syndrome (MCAP PROS). There appears to be interaction between RIT1 and PI3K-AKT because the latter pathway is needed for the growth-promoting activity of the first, at least in adenocarcinomas, but the details of this interaction are not known. If so, the PIK3CA somatic variant may not be just a chance event. It could also be of etiological relevance that Rit activation mediates resistance to cellular stress—that is, promotes cell survival. This anti-apoptotic effect could also make it more likely that a cell that spontaneously acquires a PIK3CA GoF variant will survive and proliferate. We aim to encourage clinicians to investigate atypical findings in individuals with PROS. If further similar cases are reported, this would suggest that the establishment of PROS mosaicism is facilitated by the background of a RASopathy.


2021 ◽  
Vol 7 (11) ◽  
pp. 978
Author(s):  
Gilmore T. Pambuka ◽  
Tonjock Rosemary Kinge ◽  
Soumya Ghosh ◽  
Errol D. Cason ◽  
Martin M. Nyaga ◽  
...  

Plant-associated fungi, or the mycobiome, inhabit plant surfaces above ground, reside in plant tissues as endophytes, or are rhizosphere in the narrow zone of soil surrounding plant roots. Studies have characterized mycobiomes of various plant species, but little is known about the sorghum mycobiome, especially in Africa, despite sorghum being one of the most important indigenous and commercial cereals in Africa. In this study, the mycobiome associated with above- and below-ground tissues of three commercial sorghum cultivars, as well as from rhizosphere and surrounding bulk soil samples, were sequenced using targeted sequencing with the Illumina MiSeq platform. Relative abundance differences between fungal communities were found between above-ground and below-ground niches, with most differences mostly in the dominant MOTUs, such as Davidiellaceae sp. (Cladosporium), Didymellaceae sp. 1 (Phoma), Fusarium, Cryptococcus and Mucor. Above-ground communities also appeared to be more diverse than below-ground communities, and plants harboured the most diversity. A considerable number of MOTUs were shared between the cultivars although, especially for NS5511, their abundances often differed. Several of the detected fungal groups include species that are plant pathogens of sorghum, such as Fusarium, and, at low levels, Alternaria and the Ustilaginomycetes. Findings from this study illustrate the usefulness of targeted sequencing of the ITS rDNA gene region (ITS2) to survey and monitor sorghum fungal communities and those from associated soils. This knowledge may provide tools for disease management and crop production and improvement.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 863-863
Author(s):  
Stanley R Clarke ◽  
Adrianna Vlachos ◽  
Jens Lichtenberg ◽  
Nancy E Seidel ◽  
Jaya Jagadeesh ◽  
...  

Abstract Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis. Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR. We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing. We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift. We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA. Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. Disclosures Vlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.


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