scholarly journals Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S–23S rRNA region

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Maja Kosecka-Strojek ◽  
Artur J. Sabat ◽  
Viktoria Akkerboom ◽  
Anna M. D. Kooistra-Smid ◽  
Jacek Miedzobrodzki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Analysis ◽  
16S Rrna ◽  
Species Identification ◽  
23S Rrna ◽  
Clinical Samples ◽  
Next Generation ◽  
Enterococcal Species ◽  
Reference Sequences ◽  
Generation Sequencing

Abstract Background Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region. Methods Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region. Results This study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

scholarly journals Application of Next-Generation Sequencing Technology Based on Single Gene Locus in Species Identification of Mixed Meat Products

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Xinmei Liu ◽  
Zhiyang Liu ◽  
Yiyu Cheng ◽  
Haijing Wu ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Species Identification ◽  
Growth Hormone Receptor ◽  
Single Gene ◽  
Meat Products ◽  
Quantitative Detection ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Polymerase chain reaction (PCR) detection is a commonly used method for species identification of meat products. However, this method is not suitable for the analysis of meat products containing multiple mixtures. This study aimed to test whether next-generation sequencing (NGS) technology could be used as a method for the certification of mixed meat products. In this study, five kinds of common meat (pigs, cattle, sheep, chickens, and ducks) were mixed as samples with different proportions. The primers designed from mitochondrial 16S rRNA and nuclear genome gene (growth hormone receptor, GHR), respectively, were used to detect these meats. The sequencing results of NGS were analyzed using a self-designed bioinformatics program. The fragments with similar sequences were classified and compared with the database to determine their species. The results showed that all five kinds of meat components could be correctly identified using these two primers. The meat composition could be detected as low as 0.5% in the mixed samples using the NGS technology targeting GHR gene fragments, which was superior to those targeting mitochondrial 16S rRNA. However, the quantitative detection of species in the mixture was not likely to be quite accurate due to the amplification bias of PCR amplification. These results showed that the NGS technology could be applied to identify meat species in mixtures.

scholarly journals Rapid 16S rRNA Next-Generation Sequencing of Polymicrobial Clinical Samples for Diagnosis of Complex Bacterial Infections

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e65226 ◽  
Author(s):  
Stephen J. Salipante ◽  
Dhruba J. Sengupta ◽  
Christopher Rosenthal ◽  
Gina Costa ◽  
Jessica Spangler ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Bacterial Infections ◽  
Clinical Samples ◽  
Next Generation ◽  
Generation Sequencing

Next Generation Sequencing (NGS) approach applied to species identification in mixed processed seafood products

Food Control ◽  
2021 ◽  
pp. 108590
Author(s):  
Roberta Piredda ◽  
Anna Mottola ◽  
Giulia Cipriano ◽  
Roberto Carlucci ◽  
Giuseppina Ciccarese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Species Identification ◽  
Next Generation ◽  
Seafood Products ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

scholarly journals Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

2021 ◽  
Vol 15 (10) ◽  
pp. e0009779
Author(s):  
Fakhriddin Sarzhanov ◽  
Funda Dogruman-Al ◽  
Monica Santin ◽  
Jenny G. Maloney ◽  
Ayse Semra Gureser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gastrointestinal Symptoms ◽  
Molecular Methods ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Next Generation ◽  
Dientamoeba Fragilis ◽  
Significant Difference ◽  
Generation Sequencing ◽  
Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

2019 ◽  
Vol 73 (2) ◽  
pp. 83-89 ◽  
Author(s):  
Jiuhong Pang ◽  
Tatyana Gindin ◽  
Mahesh Mansukhani ◽  
Helen Fernandes ◽  
Susan Hsiao
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Clinical Samples ◽  
Next Generation ◽  
Diverse Range ◽  
Tree Classifier ◽  
Generation Sequencing ◽  
Cancer Panel ◽  
Clinical Cases

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP).MethodsAltered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay.ResultsThe classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours.ConclusionThis study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

Next-generation sequencing for tumor mutation quantification using liquid biopsies

2020 ◽  
Vol 58 (2) ◽  
pp. 306-313 ◽  
Author(s):  
Mariano Provencio ◽  
Clara Pérez-Barrios ◽  
Miguel Barquin ◽  
Virginia Calvo ◽  
Fabio Franco ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Correlation Coefficient ◽  
Resistance Mutation ◽  
Response To Treatment ◽  
Clinical Samples ◽  
Next Generation ◽  
Liquid Biopsies ◽  
Nsclc Patients ◽  
Generation Sequencing

AbstractBackgroundNon-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations.MethodsA total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR.ResultsLin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input.ConclusionsMAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.

What Else Is in Salviae officinalis folium? Comprehensive Species Identification of Plant Raw Material by DNA Metabarcoding

Planta Medica ◽  
2017 ◽  
Vol 84 (06/07) ◽  
pp. 428-433 ◽  
Author(s):  
Corinna Schmiderer ◽  
Brigitte Lukas ◽  
Joana Ruzicka ◽  
Johannes Novak
Keyword(s):  
Next Generation Sequencing ◽  
Dna Barcoding ◽  
Species Identification ◽  
Internal Transcribed Spacer ◽  
Raw Material ◽  
Next Generation ◽  
Additional Species ◽  
Internal Transcribed Spacer 2 ◽  
Dna Metabarcoding ◽  
Generation Sequencing

AbstractQuality control of drugs consists of identifying the raw material to avoid unwanted admixtures or exchange of material as well as looking for abiotic and biotic contaminations. So far, identity and microbial contamination are analyzed by separate processes and separate methods. Species identification by their DNA (“DNA barcoding”) has the potential to supplement existing methods of identification. The introduction of next-generation sequencing methods offers completely new approaches like the identification of whole communities in one analysis, termed “DNA metabarcoding”. Here we present a next-generation sequencing assessment to identify plants and fungi of two commercial sage samples (Salvia officinalis) using the standard DNA barcoding region “internal transcribed spacer” consisting of internal transcribed spacer 1 and internal transcribed spacer 2, respectively. The main species in both samples was identified as S. officinalis. The spectrum of accompanying plant and fungal species, however, was completely different between the samples. Additionally, the composition between internal transcribed spacer 1 and internal transcribed spacer 2 within the samples was different and demonstrated the influence of primer selection and therefore the need for harmonization. This next-generation sequencing approach does not result in quantitative species composition but gives deeper insight into the composition of additional species. Therefore, it would allow for a better knowledge-based risk assessment than any other method available. However, the method is only economically feasible in routine analysis if a high sample throughput can be guaranteed.

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