BRACNAC: A and Copy Number Alteration Caller from Next-Generation Sequencing Data.

Detecting copy number variations (CNVs) and alterations (CNAs) in the and genes is essential for testing patients for targeted therapy applicability. However, the available bioinformatics tools were initially designed for identifying CNVs/CNAs in whole-genome or -exome (WES) NGS data or targeted NGS data without adaptation to the genes. Most of these tools were tested on sample cohorts of limited size, with their use restricted to specific library preparation kits or sequencing platforms.

Detecting Microsatellite Instability in Endometrial, Colon, and Stomach Cancers Using Targeted NGS.

Purpose: To develop a method for testing the MSI based on targeted NGS.

Methods: Based on the results of previous studies, 81 microsatellite loci with high variability in MSI-H tumors were selected, and a method for calculating the MSI score was developed. Using the MSI score, we defined the MSI status in endometral (162), colon (153), and stomach (190) cancers.

Identification of Chimeric NTRK3 Genes in Papillary Thyroid Cancer Cells by Analyzing the Imbalance of the Expression of 5' and 3' mRNA Fragments.

The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR).

Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase Chimeric Domain That Cause Resistance to Larotrectinib.

The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting.

Differences in Transcriptomic Profiles of Brain and Thyroid Tumors with NTRK Gene Rearrangement.

For tumors with chimeric NTRK genes, entrectinib and larotrectinib can be prescribed regardless of tumor localization. We compared changes in the transcriptional activity of genes in brain tumors (BT) and thyroid cancer (TC) with rearrangement (NTRK+) and without rearrangement (NTRK-) of the NTRK genes using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We revealed an increase in the transcription of the JUN gene in NTRK+ samples in comparison with NTRK- samples: by 1.

A spectrum of BRCA1 and BRCA2 germline deleterious variants in ovarian cancer in Russia.

Purpose: Pathogenic variants (PVs) in BRCA1 and BRCA2 genes are essential biomarkers of an increased breast and ovarian cancer risk and tumor sensitivity to poly ADP ribose polymerase inhibitors. In Russia, eight PVs were thought to be the most common, among which BRCA1 c.5266dup is the most frequently identified one.

Development of a Test System to Detect the Omicron Variant of SARS-CoV-2 and the Frequency of Its Detection in Patients.

We developed a new test system to detect the omicron variant of SARS-CoV-2 using allele-specific reverse transcription PCR and estimated the frequency of its detection in patients living in the Novosibirsk Region. Clinical samples were divided into 3 groups: samples collected from December 1 to December 30, 2021 (group 1; n=66), from December 30, 2021 to January 10, 2022 (group 2; n=20), and from January 11 to January 22, 2022 (group 3; n=101). Based on the identification of 5 mutations specific to SARS-CoV-2 (B.

The Level of LINE-1 mRNA Is Increased in Extracellular Circulating Plasma RNA in Patients with Colorectal Cancer.

We performed a comparative quantitative analysis of LINE-1 mRNA levels in extracellular total plasma RNA in patients with colon cancer and practically healthy donors. Quantitative multiplex PCR with reverse transcription was used to assess the level of LINE-1 and 18S rRNA mRNA in extracellular total plasma RNA. The median of LINE-1 mRNA values in colon cancer patients (4.

Prediction of EVT6-NTRK3-Dependent Papillary Thyroid Cancer Using Minor Expression Profile.

Solid tumors resulting from oncogenic stimulation of neurotrophin receptors (TRK) by chimeric proteins are a group of rare tumors of various localization that respond to therapy with targeted drugs entrectinib and larotrectinib. The standard method for detecting chimeric TRK genes in tumor samples today is considered to be next generation sequencing with the determination of the prime structure of the chimeric transcripts. We hypothesized that expression of the chimeric tyrosine kinase proteins in tumors can determine the specific transcriptomic profile of tumor cells.

Multiplex Droplet Digital PCR Assay for Detection of and Genes Amplification in Non-Small Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, and , are among targets for these specific therapeutic agents.

An inexpensive, simple and effective method of genome DNA fragmentation for NGS libraries.

Next-generation sequencing (NGS)-library preparation for whole-genome sequencing (WGS) starts with DNA fragmentation, and sonication is a physical approach used most often due to its simplicity and reproducibility. However, the commercially available Covaris instrument has a high price for both the device and consumables. Here, we describe our in-house method of DNA shearing by sonication with small (100-600 µm) glass beads and an ultrasonic bath.

NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions.

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels.

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase.

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii.

Spectrum of Mutations in BRCA1/2 Associated High-Grade Serous Ovarian Cancer.

Mutations in TP53 lead to loss of function (LOF) or gain of function (GOF) of the corresponding protein p53 and produce a different effect on the tumor. Our goal was to determine the spectrum of somatic variants in associated high-grade serous ovarian cancer (HGSOC). The population under study comprised of HGSOCs with pathogenic variants in ( = 78) or ( = 21).

Multiplex ddPCR assay for screening copy number variations in BRCA1 gene.

Purpose: Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene.

Methods: In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status.

Walking pathways with positive feedback loops reveal DNA methylation biomarkers of colorectal cancer.

Background: The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes.

BRCA-analyzer: Automatic workflow for processing NGS reads of BRCA1 and BRCA2 genes.

The use of targeted next-generation sequencing (NGS) provides great new opportunities for molecular and medical genetics. However, in order to take advantage of these opportunities, we need to have reliable tools for extracting the necessary information from the huge amount of data generated by NGS. Here we present our automatic multithreaded workflow for processing NGS data of BRCA1 and BRCA2 genes obtained with NGS technology named BRCA-analyzer.

One-phase phenol-free method for microRNA isolation from blood plasma.

MicroRNA extraction is an essential procedure when discovering MicroRNA-based biomarkers and approaches. Here we describe a new method for microRNA isolation from human blood plasma, based on isopropanol precipitation from the one-phase lysate. We demonstrate that the use of more than four volumes of lysis buffer based on 5 M guanidine isothiocyanate prevents the formation of large, viscous, and hardly soluble precipitate.

Loss of Heterozygosity in BRCA1 and BRCA2 Genes in Patients with Ovarian Cancer and Probability of Its Use for Clinical Classification of Variations.

Changes (or variants) in BRCA1 and BRCA2 gene sequences can have different lengths and clinical significance: from single nucleotide variants (SNV) and short insertions/deletions (<50 bp) to extended deletions and duplications (so-called copy number variations, or CNV). According to their clinical significance, all variants can be divided into pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign. Moreover, variants can be germinal (i.

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype.

Purpose: MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection.

Computational master-regulator search reveals mTOR and PI3K pathways responsible for low sensitivity of NCI-H292 and A427 lung cancer cell lines to cytotoxic action of p53 activator Nutlin-3.

Background: Small molecule Nutlin-3 reactivates p53 in cancer cells by interacting with the complex between p53 and its repressor Mdm-2 and causing an increase in cancer cell apoptosis. Therefore, Nutlin-3 has potent anticancer properties. Clinical and experimental studies of Nutlin-3 showed that some cancer cells may lose sensitivity to this compound.

Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance.

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp.

cutPrimers: A New Tool for Accurate Cutting of Primers from Reads of Targeted Next Generation Sequencing.

Cutting of primers from reads is an important step of processing targeted amplicon-based next generation sequencing data. Existing tools are adapted for cutting of one or several primer/adapter sequences from reads and removing all of their occurrences. Also most of the existing tools use kmers and may cut only part of primers or primers with studied sequence of gene.

Highly Sensitive and Reliable Detection of EGFR Exon 19 Deletions by Droplet Digital Polymerase Chain Reaction.

Background: Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined.

Methods: We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19.

Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies.