95 research outputs found
A mutate-and-map protocol for inferring base pairs in structured RNA
Chemical mapping is a widespread technique for structural analysis of nucleic
acids in which a molecule's reactivity to different probes is quantified at
single-nucleotide resolution and used to constrain structural modeling. This
experimental framework has been extensively revisited in the past decade with
new strategies for high-throughput read-outs, chemical modification, and rapid
data analysis. Recently, we have coupled the technique to high-throughput
mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure
of not only that nucleotide but also its interaction partner. Carrying out the
mutation and mapping for the entire system gives an experimental approximation
of the molecules contact map. Here, we give our in-house protocol for this
mutate-and-map strategy, based on 96-well capillary electrophoresis, and we
provide practical tips on interpreting the data to infer nucleic acid
structure.Comment: 22 pages, 5 figure
Whole-exome sequencing in patients with protein aggregate myopathies reveals causative mutations associated with novel atypical phenotypes
BACKGROUND: Myofibrillar myopathies (MFM) are a subgroup of protein aggregate myopathies (PAM) characterized by a common histological picture of myofibrillar dissolution, Z-disk disintegration, and accumulation of degradation products into inclusions. Mutations in genes encoding components of the Z-disk or Z-disk-associated proteins occur in some patients whereas in most of the cases, the causative gene defect is still unknown. We aimed to search for pathogenic mutations in genes not previously associated with MFM phenotype.METHODS: We performed whole-exome sequencing in four patients from three unrelated families who were diagnosed with PAM without aberrations in causative genes for MFM.RESULTS: In the first patient and her affected daughter, we identified a heterozygous p.(Arg89Cys) missense mutation in LMNA gene which has not been linked with PAM pathology before. In the second patient, a heterozygous p.(Asn4807Phe) mutation in RYR1 not previously described in PAM represents a novel, candidate gene with a possible causative role in the disease. Finally, in the third patient and his symptomatic daughter, we found a previously reported heterozygous p.(Cys30071Arg) mutation in TTN gene that was clinically associated with cardiac involvement.CONCLUSIONS: Our study identifies a new genetic background in PAM pathology and expands the clinical phenotype of known pathogenic mutations
Polymorphisms of the DNA repair genes XRCC1 and ERCC4 are not associated with smoking- and drinking-dependent larynx cancer in a polish population
Aim of this article is to investigate the association between the genotypes of the XRCC1-Arg399Gln (rs25487) and ERCC4-Arg415Gln (rs1800067) polymorphisms and smoking- and drinking-related larynx cancer in a Polish population
Genomic landscape of human erythroleukemia K562 cell line, as determined by next-generation sequencing and cytogenetics
We have performed detailed analysis of the genomic landscape of commercially available K562 cells, employing targeted enrichment of nearly 1300 cancer-related genes followed by next-generation sequencing (NGS) and also classical cytogenetics. Deep sequencing revealed 88 variants of potentially biological significance. Among them we have detected alterations in genes already known to be mutated in K562, such as TP53 but also in several other genes, which are implicated in tumorigenesis and drug resistance, such as MLH1, ASXL1 and BRCA1 as the most prominent examples. Fluorescence in situ hybridization (FISH) of interphases of K562 cells revealed multiplication of the BCR and ABL1 gene copies, as well as the amplification of the BCR-ABL1 fusion gene. Our results may help to better understand genomic instability of the blastic phase of CML represented by the K562 cell line and can help researchers who want to employ this cell line in various experimental settings
Next-generation sequencing for diagnosis of thoracic aortic aneurysms and dissections: diagnostic yield, novel mutations and genotype phenotype correlations
Additional file 1. Complementary data on primer sequences, SKI amplification and survival analyses
Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta
Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina pairedend, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The fnal genome assembly is 177Mbp with contig N50 size of 75kbp and a scafold N50 size of 2.3Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts
First familial cases of type 2 congenital erythrocytosis (ECYT2) with a Chuvash pathogenic variant in gene in Poland: example of the clinical utility of next-generation sequencing in diagnostics of orphan diseases
In this article, we report familial cases of type 2 congenital erythrocytosis (ECYT2) in two siblings, a 2-year-old boy and his younger sister. Both patients were diagnosed based on laboratory findings including erythrocytosis, elevated hemoglobin levels, and hematocrit. Acquired erythrocytosis was excluded based on the clinical features and genetic analysis of genes. Next-generation sequencing was employed for older brother revealing NM_000551.4: c.598C>T, p.Arg200Trp homozygous variant in the gene, the similar variant was detected in the younger sibling. Sequencing analysis confirmed the c.598C>T heterozygous variant in both parents. To the best of our knowledge, these are the first confirmed cases of familial erythrocytosis type 2, also known as Chuvash type, in Poland
Differences in the composition of the bacterial element of the urinary tract microbiome in patients undergoing dialysis and patients after kidney transplantation
IntroductionThe development of molecular biology methods and their application in microbial research allowed the detection of many new pathogens that cause urinary tract infections (UTIs). Despite the advances of using new research techniques, the etiopathogenesis of UTIs, especially in patients undergoing dialysis and patients after kidney transplantation, is still not fully understood.MethodsThis study aimed to characterize and compare the composition of the bacterial element of the urinary tract microbiome between the groups of patients undergoing dialysis (n = 50) and patients after kidney transplantation (n = 50), with positive or negative urine culture, compared to healthy individuals (n = 50).ResultsAsymptomatic bacteriuria was observed in 30% of the urine cultures of patients undergoing dialysis and patients after kidney transplantation, with Escherichia coli as the most dominant microorganism (73%) detected with the use of classical microbiology techniques. However, differences in the bacterial composition of the urine samples between the evaluated patient groups were demonstrated using the amplicon sequencing. Finegoldia, Leptotrichia, and Corynebacterium were found to be discriminative bacteria genera in patients after dialysis and kidney transplantation compared to the control group. In addition, in all of urine samples, including those without bacteriuria in classical urine culture, many types of bacteria have been identified using 16S rRNA sequencing.DiscussionThe revealed microbial characteristics may form the basis in searching for new diagnostic markers in treatment of patients undergoing dialysis and patients after kidney transplantation
- …