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Query: "keywords" (next-generation sequencing) .

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1.
Extreme environments simplify reassembly of communities of arbuscular mycorrhizal fungi
Nataša Šibanc, Dave R. Clark, Thorunn Helgason, Alex J. Dumbrell, Irena Maček, 2024, original scientific article

Abstract: The ecological impacts of long-term (press) disturbance on mechanisms regulating the relative abundance (i.e., commonness or rarity) and temporal dynamics of species within a community remain largely unknown. This is particularly true for the functionally important arbuscular mycorrhizal (AM) fungi; obligate plant-root endosymbionts that colonize more than two-thirds of terrestrial plant species. Here, we use high-resolution amplicon sequencing to examine how AM fungal communities in a specific extreme ecosystem—mofettes or natural CO2 springs caused by geological CO2 exhalations—are affected by long-term stress. We found that in mofettes, specific and temporally stable communities form as a subset of the local metacommunity. These communities are less diverse and dominated by adapted, “stress tolerant” taxa. Those taxa are rare in control locations and more benign environments worldwide, but show a stable temporal pattern in the extreme sites, consistently dominating the communities in grassland mofettes. This pattern of lower diversity and high dominance of specific taxa has been confirmed as relatively stable over several sampling years and is independently observed across multiple geographic locations (mofettes in different countries). This study implies that the response of soil microbial community composition to long-term stress is relatively predictable, which can also reflect the community response to other anthropogenic stressors (e.g., heavy metal pollution or land use change). Moreover, as AM fungi are functionally differentiated, with different taxa providing different benefits to host plants, changes in community structure in response to long-term environmental change have the potential to impact terrestrial plant communities and their productivity
Keywords: arbuscular mycorrhiza, elevated CO2, long-term experiments, soil biodiversity, soil hypoxia, next-generation sequencing, NGS
Published in DiRROS: 28.02.2024; Views: 97; Downloads: 49
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2.
The influence of storage conditions and DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.)
Zina Devetak, Andreja Kavčič, Maarten De Groot, Barbara Piškur, 2023, original scientific article

Abstract: One of the key steps of the molecular identification of bark beetles is obtaining a sufficient quantity of high-quality DNA extract. In this study, we investigated the influence of different storage procedures for Ips typographus (L.) specimens and various DNA extraction protocols on the quantity and quality of DNA intended for use in molecular diagnostics. Adult beetles were frozen at -20 °C, either dry or in ethanol. We tested four different protocols for DNA extraction. We compared the quantity of extracted DNA and assessed its quality with PCR and Sanger sequencing. Different storage protocols had no significant effect on the quantity of DNA extracted. However, freezing specimens in ethanol provided higher-quality DNA for molecular applications. Only two of the extraction protocols produced sequenceable amplicons, and the difference in the amount of extracted DNA between them was not significant. We propose the optimal combination of storing specimens in ethanol at -20°C and using the Nucleospin Insect DNA extraction kit from Macherey Nagel, enabling a timeefficient identification process.
Keywords: early detection, specimen storage, total DNA extraction, PCR, polymerase chain reaction, Sanger sequencing, molecular diagnostics
Published in DiRROS: 02.02.2024; Views: 331; Downloads: 101
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3.
New approach for detection of normal alternative splicing events and aberrant spliceogenic transcripts with long-range PCR and deep RNA sequencing
Vita Šetrajčič Dragoš, Vida Stegel, Ana Blatnik, Gašper Klančar, Mateja Krajc, Srdjan Novaković, 2021, original scientific article

Abstract: RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method%s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant%s impact on splicing.
Keywords: RNA sequencing, DNA variant, splicing
Published in DiRROS: 21.09.2022; Views: 416; Downloads: 235
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4.
Identification of spliceogenic variants beyond canonical GT-AG splice sites in hereditary cancer genes
Vita Šetrajčič Dragoš, Ksenija Strojnik, Gašper Klančar, Petra Škerl, Vida Stegel, Ana Blatnik, Marta Banjac, Mateja Krajc, Srdjan Novaković, 2022, original scientific article

Abstract: Pathogenic/likely pathogenic variants in susceptibility genes that interrupt RNA splicing are a well-documented mechanism of hereditary cancer syndromes development. However, if RNA studies are not performed, most of the variants beyond the canonical GT-AG splice site are characterized as variants of uncertain significance (VUS). To decrease the VUS burden, we have bioinformatically evaluated all novel VUS detected in 732 consecutive patients tested in the routine genetic counseling process. Twelve VUS that were predicted to cause splicing defects were selected for mRNA analysis. Here, we report a functional characterization of 12 variants located beyond the first two intronic nucleotides using RNAseq in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes. Based on the analysis of mRNA, we have successfully reclassified 50% of investigated variants. 25% of variants were downgraded to likely benign, whereas 25% were upgraded to likely pathogenic leading to improved clinical management of the patient and the family members.
Keywords: hereditary cancer, RNA sequencing, spliceogenic
Published in DiRROS: 07.09.2022; Views: 445; Downloads: 231
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5.
The Carniolan honeybee from Slovenia : a complete and annotated mitochondrial genome with comparisons to closely related Apis mellifera subspecies
Ajda Moškrič, Andraž Marinč, Polonca Ferk, Branimir Leskošek, Mai-Britt Mosbech, Ignas Bunikis, Olga Vinnere Pettersson, Lucile Soler, Janez Prešern, original scientific article

Abstract: The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera—a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies’ conservation and preservation as well as its breeding and selection.
Keywords: western honeybee, Apis mellifera carnica, mitochondrial genome, phylogeny, C lineage, annotation, whole genome sequencing, protection, conservation
Published in DiRROS: 06.09.2022; Views: 476; Downloads: 174
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6.
Using genomic information for management planning of an endangered perennial, Viola uliginosa
Kyung Min Lee, Pertti Ranta, Jarmo Saarikivi, Lado Kutnar, Branko Vreš, Maxim Dzhus, Marko Mutanen, Laura Kvist, 2020, original scientific article

Abstract: Species occupying habitats subjected to frequent natural and/or anthropogenic changes are a challenge for conservation management. We studied one such species, Viola uliginosa, an endangered perennial wetland species typically inhabiting sporadi-cally flooded meadows alongside rivers/lakes. In order to estimate genomic diversity, population structure, and history, we sampled five sites in Finland, three in Estonia, and one each in Slovenia, Belarus, and Poland using genomic SNP data with double-digest restriction site-associated DNA sequencing (ddRAD-seq). We found mono-phyletic populations, high levels of inbreeding (mean population FSNP = 0.407-0.945), low effective population sizes (Ne = 0.8-50.9), indications of past demographic ex-pansion, and rare long-distance dispersal. Our results are important in implementing conservation strategies for V. uliginosa, which should include founding of seed banks, ex situ cultivations, and reintroductions with individuals of proper origin, combined with continuous population monitoring and habitat management.
Keywords: nature conservation, demography, genomic diversity, population genomics, RAD sequencing, Viola
Published in DiRROS: 20.01.2022; Views: 800; Downloads: 646
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7.
Next-generation sequencing to characterize pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries
Eva Sodja, Simon Koren, Nataša Toplak, Sara Truden, Marija Žolnir-Dovč, 2021, original scientific article

Abstract: Objectives. Next-generation sequencing (NGS) provide a comprehensive analysis of the genetic alterations that are most commonly linked with pyrazinamide (PZA) resistance. There are no studies reporting molecular background of PZA resistance in TB isolates from Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance, pncA, using Ion Torrent technology in comparison to phenotypic BACTEC MGIT 960 DST in clinical TB isolates from two countries of the Balkan Peninsula. Methods. Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using the AmpliSeq™ Kit for Chef DL8 and the Ion AmpliSeq TB Research Panel. Result.s Of all 61 TB isolates included, 56 TB were phenotypically resistant to any antibiotic. Among them, 38/56 (67.9%) TB isolates were phenotypically resistant to pyrazinamide and pncA mutations were detected in 33/38 cases (86.8%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS to conventional BACTEC MGIT 960 DST revealed very strong agreement (90.2%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Conclusions. Detection of PZA resistance using NGS seems to be a valuable tool for surveillance of TB drug resistance also in the Balkan Peninsula, with great potential to provide useful information at least one weak earlier than is possible with phenotypic DST.
Keywords: tuberculosis, Mycobacterium tuberculosis, high-throughput nucleotide sequencing, pyrazinamide, microbial sensitivity tests, next-generation sequencing, drug susceptibility testing, Slovenia, Republic of North Macedonia
Published in DiRROS: 10.01.2022; Views: 856; Downloads: 524
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8.
Genomic insights into the Mycobacterium kansasii complex : an update
Tomasz Jagielski, Paulina Borówka, Zofia Bakuła, Jakub Lach, Błażej Marciniak, Anna Brzostek, Jarosław Dziadek, Mikołaj Dziurzyński, Lian Pennings, Jakobus van Ingen, Marija Žolnir-Dovč, Dominik Strapagiel, 2020, original scientific article

Abstract: Only very recently, has it been proposed that the hitherto existing Mycobacteriumkansasii subtypes (I–VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, amonophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.
Keywords: Mycobacteriumkansasii complex, Mycobacteriumostraviense sp. nov., non-tuberculous mycobacteria (NTM), whole genome sequencing, taxonomy
Published in DiRROS: 31.07.2020; Views: 1846; Downloads: 1317
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9.
Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries
Eva Sodja, Nataša Toplak, Simon Koren, Minka Kovač, Sara Truden, Biljana Ilievska Poposka, Marija Žolnir-Dovč, 2019, original scientific article

Abstract: Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.
Keywords: drug resistant tuberculosis, next-generation sequencing, low-incidence countries, phenotypic drug susceptibility testing
Published in DiRROS: 24.07.2020; Views: 1820; Downloads: 1024
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