First whole genome sequencing of Russian isolate of Capnocytophaga canimorsus, opportunistic pathogen causing lethal sepsis.

Capnocytophaga canimorsus is a part of healthy oral flora of dogs and cats. However, when it is transmitted to human subjects via animal bites or scratches, it can cause severe complications like endocarditis or even lethal septic shock, especially in immunocompromised persons. In this study, we performed the first whole-genome sequencing on Illumina HiSeq platform of Russian isolate of C. canimorsus that have caused lethal sepsis in 51-old male from Moscow. We believe that the availability of genomic sequence and annotation for the given strain could be useful for future epidemiological surveillance studies in Russia and other countries.

MAGERI: Computational pipeline for molecular-barcoded targeted resequencing.

Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.

Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries.

BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

Towards error-free profiling of immune repertoires.

Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

Clonal diversity, antimicrobial resistance, and genome features among nonfermenting gram-negative bacteria isolated from patients with cystic fibrosis in Russia.

Nonfermenting gram-negative (NFGN) bacteria were isolated from cystic fibrosis (CF) patients and subjected to susceptibility testing and whole-genome sequencing. Among 170 enrolled CF patients, 112 (65.9%) were colonized with at least 1 key NFGN species. The species-specific infection rate was highest for Pseudomonas aeruginosa (40.6%) followed by Stenotrophomonas maltophilia (14.1%), Achromobacter spp. (9.4%), and Burkholderia cepacia complex (Bcc, 8.2%) demonstrating a significant age-dependent increase for P. aeruginosa and Achromobacter spp., but not for S. maltophilia or Bcc. P. aeruginosa sequence types (STs) related to high-risk epidemic and global CF clones were carried by 12 (7.1%) and 13 (7.6%) patients, respectively. In total, 47% NFGN isolates, predominantly P. aeruginosa, harbored at least 1 plasmid-borne resistance gene; 5 ST235 isolates carried blaVIM2. Pathogenicity island-borne virulence genes were harbored by 9% NFGN isolates. These findings in conjunction with frequent early colonization by Bcc raised serious concerns regarding infection control in Russian CF centers.

Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms.

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

Antimicrobial Resistance and Genomic Characterization of OXA-48- and CTX-M-15-Co-Producing Hypervirulent Klebsiella pneumoniae ST23 Recovered from Nosocomial Outbreak.

Multidrug resistance (MDR) and hypervirulence (hv) have been long considered distinct evolutionary traits for Klebsiella pneumoniae (Kp), a versatile human pathogen. The recent emergence of Kp strains combining these traits poses a serious global threat. In this article, we describe the phenotypic and genomic characteristics of an MDR hvKp isolate, MAR14-456, representative of a nosocomial outbreak in Moscow, Russia, that was recovered from a postoperative wound in a patient who later developed multiple abscesses, fatal sepsis, and septic shock. Broth microdilution testing revealed decreased susceptibility of MAR14-456 to carbapenems (MICs 0.5-2 mg/L) and a high-level resistance to most ß-lactams, ß-lactam-ß-lactamase-inhibitor combinations, and non-ß-lactam antibiotics, except ceftazidime-avibactam, amikacin, tigecycline, and colistin. Whole-genome sequencing using Illumina MiSeq and ONT MinION systems allowed to identify and completely assemble two conjugative resistance plasmids, a typical 'European' epidemic IncL/M plasmid that carries the gene of OXA-48 carbapenemase, and an IncFIIK plasmid that carries the gene of CTX-M-15 ESBL and other resistance genes. MLST profile, capsular, lipopolysaccharide, virulence genes encoded on chromosome and IncHI1B/FIB plasmid, and the presence of apparently functional type I-E* CRISPR-Cas system were all characteristic of hvKp ST23, serotype K1-O1v2. Phylogenetic analysis showed the closest relatedness of MAR14-456 to ST23 isolates from China. This report highlights the threat of multiple resistance acquisition by hvKp strain and its spread as a nosocomial pathogen.

Molecular Typing, Characterization of Antimicrobial Resistance, Virulence Profiling and Analysis of Whole-Genome Sequence of Clinical Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae is one of the most important pathogens concerned with multidrug resistance in healthcare-associated infections. The treating of infections caused by this bacterium is complicated due to the emergence and rapid spreading of carbapenem-resistant strains, which are associated with high mortality rates. Recently, several hypervirulent and carbapenemase-producing isolates were reported that make the situation even more complicated. In order to better understand the resistance and virulence mechanisms, and, in turn, to develop effective treatment strategies for the infections caused by multidrug-resistant K. pneumoniae, more comprehensive genomic and phenotypic data are required. Here, we present the first detailed molecular epidemiology report based on second and third generation (long-read) sequencing for the clinical isolates of K. pneumoniae in the Russian Federation. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as the virulence and plasmid profiling for 36 K. pneumoniae isolates. We have revealed 2 new multilocus sequence typing (MLST)-based sequence types, 32 multidrug-resistant (MDR) isolates and 5 colistin-resistant isolates in our samples. Three MDR isolates belonged to a very rare ST377 type. The whole genome sequences and additional data obtained will greatly facilitate further investigations in the field of antimicrobial resistance studies.

Identification of a novel mutation in DKC1 in dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare congenital syndrome characterized by the triad of reticular skin pigmentation, nail dystrophy and mucosal leukoplakia, and the predisposition to bone marrow failure and malignancy. DC is genetically heterogeneous and X-linked and autosomal forms of the disease exist. Here, we report the clinical description and mutation analysis of a Russian family with X-linked DC. A novel mutation in DKC1 raised de novo in the maternal grandmother's gamete was found; this mutation is a 2 bp inversion in exon 3: NM_001363:c.166_167invCT (Leu56Ser).

Inactivation of the oprD porin gene by a novel insertion sequence ISPa195 associated with large deletion in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

OBJECTIVES: Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD. METHODS: Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. RESULTS: Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC=32mg/L) and meropenem (MIC=16mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected. CONCLUSION: The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux.

A high-throughput assay for quantitative measurement of PCR errors.

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.