Early infantile epileptic encephalopathies (EIEE) are a group of disorders characterized by pharmacoresistant epileptic seizures manifesting in infancy and leading to psychomotor retardation. The most common genetic variant with X-linked dominant inheritance is type 2 EIEE associated with CDKL5 gene mutations. We evaluated the prevalence of this type of EIEE among Russian patients (n = 148) with epileptic seizures manifesting in infancy and analyzed their clinical and genetic characteristics. We performed exome sequencing for all patients; 15 (10 %) of them (aged between 2 months and 5 years) were found to have CDKL5 gene mutations and were, therefore, diagnosed with type 2 EIEE. The results of correlation analysis suggest that the severity of clinical manifestations of type 2 EIEE is largely determined by the location of mutations affecting the function of the protein encoded by this gene. This is important to ensure better understanding of type 2 EIEE etiology and predict it severity in patients with different allelic variants.

BackgroundCutis laxa (CL) is a rare connective tissue disorder characterized by loose, redundant, inelastic and wrinkled skin. Patients develop a prematurely aged appearance. Inheritance can be autosomal dominant or autosomal recessive. The X-linked form is now classified in the group of copper transport diseases. Autosomal dominant CL is characterized by wrinkled, redundant and sagging, inelastic skin and in some cases is associated with internal organ involvement.Case presentationWe report a familial case of autosomal dominant CL, which includes a 33-year-old woman and her 11-year-old son with dry, thin and wrinkled skin that appeared prematurely aged. No serious involvement of internal organs was found. In both patients, we identified novel heterozygous mutation c.2323delG (p.Ala775fs) in exon 34 of elastin transcript NM_001278939.1. Similar frameshift mutations in the last exons of elastin gene were previously reported in patients with autosomal dominant CL.ConclusionsOur results show a novel frameshift mutation that was found in patients with cutis laxa. Exome sequencing is effective and useful technology for properly diagnosis of diseases with similar phenotype to ensure proper treatment is provided.

Congenital core myopathies are a clinically and genetically heterogenous group of congenital myopathies that share a specific histopathological feature: areas of reduced oxidative activity in muscle fibers. The relationship between clinical, genetic and morphological characteristics of this group of disorders remains understudied. The aim of this work was to compare clinical presentations and morphological phenotypes of patients with congenital myopathies/myodystrophy to the data yielded by massively parallel exome sequencing. Eight children were included in the study: 2 boys and 6 girls aged 3 to 14 years. Their biopsy material was analyzed by light and electron microscopy. Sequencing was performed on HiSeq2500. Mutations were detected in 7 (87.5%) of 8 participants. Six children had 8 mutations in the genes associated with congenital core myopathies; one patient had 2 mutations in the LAMA2 gene implicated in merosin-deficient muscular dystrophy. The proportions of patients with mutations in RYR1 and SEPN1 were equal (42.86%). Of 10 detected mutations, 3 had not been previously described. including c.7561G>A in RYR1, c.485C>A in SEPN1 and p.Cys1136Arg in LAMA2. The clinical and morphological features of core myopathies suggest that genetic causes of this group of disorders should not be limited to RYR1 and SEPN1 genes only. This necessitates the search for and the study of other genes implicated in congenital myopathies or myodystrophy using state-of-the-art molecular genetic tools.

Accurate library quantification is very important during post-pooling captured target sequencing. There are a number of methods available to quantify libraries prior to sequencing, but no gold standard for the quantification of libraries exists. In this study, we compared common library quantification methods (Labchip, Qubit 3.0, qPCR with three primer sets) with ultra-low coverage sequencing (MiSeq with and without insert size correction). Cost, time and quantification accuracy were considered. We found that Qubit and MiSeq were better than qPCR and LabChip at predicting the final concentration. Also we revealed that MiSeq with insert size correction was the most accurate method for library quantification prior to target sequencing. This method allows for correction shifts in the ratio due to enrichment. Ultra-low coverage sequencing by Illumina MiSeq is the most accurate method for library quantification prior to pooling and post-pooling target enrichment.

Breast cancer is one of the most widespread forms of solid tumors. By analyzing the traits of breast cancer pathogenesis at the molecular level using modern genetic analysis techniques and at different stages of the disease new data can be obtained to be further utilized in clinical practice. Molecular profiling based on next-generation sequencing is being increasingly applied as a clinical test to select target drugs for treating breast cancer patients with tumors highly resistant to therapy. In this study, we performed targeted sequencing of BRCA1 and BRCA2 oncogenes. In the total of 66 DNA samples from patients with breast tumors, BRCA1/2 mutations were found in 39 patients. There were 78 unique genetic variants, including 30 mutations in BRCA1 and 48 mutations in BRCA2. We identified 33 mutations affecting the sites of post-translational modification in proteins (PMT mutations).

Metagenomic sequencing is widely used in both scientific research and clinical practice for characterization of taxonomic profiles including estimation of relative abundance of prokaryotes in microbial communities in various media. Metagenomic sequencing of single marker genes is an excellent tool for studying the human microbiome. Unlike whole-genome sequencing, it targets those genome regions that can be instrumental in identification of microorganism species and genus. The 16S ribosomal RNA (16S rRNA) gene sequence is highly conserved but at the same time there are regions containing species-specific sequences that can discriminate between different bacteria and archaea. These regions can be amplified using universal primers, which makes the whole procedure more cost-effective and less time-consuming. Good primers and protocol design for PCR at the step of library preparation is crucial for achieving high data accuracy. Below we describe how to choose the optimal PCR protocol and universal primers to amplify V3 and V4 regions of the 16S rRNA gene for further sample sequencing using Illumnia platform.

Today, next generation sequencing (NGS) is extensively used in the research setting. However, high costs of NGS testing still prevent its routine use in clinical practice. One of the factors affecting the cost of sequencing is the number of reads per site, i.e. the number of times each nucleotide gets sequenced. On the one hand, lower coverage makes the whole process much faster and less time-consuming. On the other hand, it results in poor data quality. No unanimous opinion has been reached yet as to what minimum depth of coverage can produce reliable results. The aim of this study was to determine the minimum number of reads sufficient for accurate base calling of heterozygous and single nucleotide variants (SNV). Using bioinformatics methods, we demonstrate that accuracy can be achieved at a minimum depth of 12X.

We aimed to develop a pipeline for the bioinformatic analysis and interpretation of NGS data and detection of a wide range of single-nucleotide somatic mutations within tumor DNA. Initially, the NGS reads were submitted to a quality control check by the Cutadapt program. Low-quality 3¢-nucleotides were removed. After that the reads were mapped to the reference genome hg19 (GRCh37.p13) by BWA. The SAMtools program was used for exclusion of duplicates. MuTect was used for SNV calling. The functional effect of SNVs was evaluated using the algorithm, including annotation and evaluation of SNV pathogenicity by SnpEff and analysis of such databases as COSMIC, dbNSFP, Clinvar, and OMIM. The effect of SNV on the protein function was estimated by SIFT and PolyPhen2. Mutation frequencies were obtained from 1000 Genomes and ExAC projects, as well as from our own databases with frequency data. In order to evaluate the pipeline we used 18 breast cancer tumor biopsies. The MYbaits Onconome KL v1.5 Panel (“MYcroarray”) was used for targeted enrichment. NGS was performed on the Illumina HiSeq 2500 platform. As a result, we identified alterations in BRCA1, BRCA2, ATM, CDH1, CHEK2, TP53 genes that affected the sequence of encoded proteins. Our pipeline can be used for effective search and annotation of tumor SNVs. In this study, for the first time, we have tested this pipeline for NGS data analysis of samples from patients of the Russian population. However, further confirmation of efficiency and accuracy of the pipeline is required on NGS data from larger datasets as well as data from several types of solid tumors.

Breast cancer (BC) is one of the most common malignancies. There is a need for novel approaches to screening for genetic mutations in patients with BC that will help to reduce high mortality rates caused by this disease and improve treatment outcomes. In this study we employed next generation sequencing to screen a few key genes associated with the risk of breast cancer for mutations. We also evaluated their pathogenicity using the previously proposed bioinformatics-based algorithm and analyzed the associations between some of the detected mutations and the clinical manifestations of the disease. Our study recruited 16 female patients with BC (mean age was 50.7 +/- 11.3 years). A total of 58 mutations were detected in the oncogenes BRCA1, BRCA2, ATM, CDH1, CHEK2 and TP53. Bioinformatic analysis of the sequencing data revealed 14 mutations that affect the sequence of the encoded proteins. Most deleterious mutations were harbored by the genes BRCA1/2, ATM and TP53.