scholarly journals Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

2015 ◽  
Vol 53 (12) ◽  
pp. 3779-3783 ◽  
Author(s):  
Nontuthuko E. Maningi ◽  
Luke T. Daum ◽  
John D. Rodriguez ◽  
Matsie Mphahlele ◽  
Remco P. H. Peters ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Detection Sensitivity ◽  
Next Generation ◽  
Resistance Mutations ◽  
Coding Region ◽  
Content Type ◽  
Mdr Tb ◽  
Generation Sequencing

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance inMycobacterium tuberculosisisolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising thepncAcoding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-typepncAgene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n= 88), and concordance values between the Bactec 460, the Wayne test, orpncAgene Sanger sequencing and NGS results were 82% (n= 88), 83% (n= 88), and 89% (n= 88), respectively. NGS confirmed the majority ofpncAmutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates,pncAmutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS ofpncAimproved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

scholarly journals Search for Novel Candidate Mutations for Metronidazole Resistance in Helicobacter pylori Using Next-Generation Sequencing

2015 ◽  
Vol 59 (4) ◽  
pp. 2343-2348 ◽  
Author(s):  
Tran Thanh Binh ◽  
Rumiko Suzuki ◽  
Tran Thi Huyen Trang ◽  
Dong Hyeon Kwon ◽  
Yoshio Yamaoka
Keyword(s):  
Helicobacter Pylori ◽  
Next Generation Sequencing ◽  
Single Mutation ◽  
Next Generation ◽  
Resistance Mutations ◽  
Content Type ◽  
Metronidazole Resistance ◽  
Key Factor ◽  
H Pylori ◽  
Generation Sequencing

ABSTRACTMetronidazole resistance is a key factor associated withHelicobacter pyloritreatment failure. Although this resistance is mainly associated with mutations in therdxAandfrxAgenes, the question of whether metronidazole resistance is caused by the inactivation offrxAalone is still debated. Furthermore, it is unclear whether there are other mutations involved in addition to the two genes that are associated with resistance. A metronidazole-resistant strain was cultured from the metronidazole-susceptibleH. pyloristrain 26695-1 by exposure to low concentrations of metronidazole. The genome sequences of both susceptible and resistantH. pyloristrains were determined by Illumina next-generation sequencing, from which putative candidate resistance mutations were identified. Natural transformation was used to introduce PCR products containing candidate mutations into the susceptible parent strain 26695-1, and the metronidazole MIC was determined for each strain. Mutations infrxA(hp0642),rdxA(hp0954), andrpsU(hp0562) were confirmed by the Sanger method. The mutated sequence inrdxAwas successfully transformed into strain 26695-1, and the transformants showed resistance to metronidazole. The transformants containing a single mutation inrdxAshowed a low MIC (16 mg/liter), while those containing mutations in bothrdxAandfrxAshowed a higher MIC (48 mg/liter). No transformants containing a single mutation infrxAorrpsUwere obtained. Next-generation sequencing was used to identify mutations related to drug resistance. We confirmed that the mutations inrdxAare mainly associated with metronidazole resistance, and mutations infrxAare able to enhanceH. pyloriresistance only in the presence ofrdxAmutations. Moreover, mutations inrpsUmay play a role in metronidazole resistance.

scholarly journals Assessing Nanopore Sequencing for Clinical Diagnostics: a Comparison of Next-Generation Sequencing (NGS) Methods for Mycobacterium tuberculosis

2020 ◽  
Vol 59 (1) ◽  
pp. e00583-20
Author(s):  
Carol Smith ◽  
Tanya A. Halse ◽  
Joseph Shea ◽  
Herns Modestil ◽  
Randal C. Fowler ◽  
...  
Keyword(s):  
Public Health ◽  
Mycobacterium Tuberculosis ◽  
Next Generation Sequencing ◽  
Illumina Miseq ◽  
Drug Susceptibility ◽  
Clinical Diagnostics ◽  
Nanopore Sequencing ◽  
Next Generation ◽  
Content Type ◽  
Generation Sequencing

ABSTRACTNext-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.

Identification of minority resistance mutations in the HIV-1 integrase coding region using next generation sequencing

2015 ◽  
Vol 73 ◽  
pp. 95-100 ◽  
Author(s):  
Jannik Fonager ◽  
Jonas T. Larsson ◽  
Christian Hussing ◽  
Frederik Neess Engsig ◽  
Claus Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Resistance Mutations ◽  
Coding Region ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

2018 ◽  
Vol 15 (8) ◽  
pp. 1697 ◽  
Author(s):  
Andrea Arias ◽  
Pablo López ◽  
Raphael Sánchez ◽  
Yasuhiro Yamamura ◽  
Vanessa Rivera-Amill
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
T Cell ◽  
Sanger Sequencing ◽  
Response To Therapy ◽  
Next Generation ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Generation Sequencing ◽  
Hiv 1

The implementation of antiretroviral treatment combined with the monitoring of drug resistance mutations improves the quality of life of HIV-1 positive patients. The drug resistance mutation patterns and viral genotypes are currently analyzed by DNA sequencing of the virus in the plasma of patients. However, the virus compartmentalizes, and different T cell subsets may harbor distinct viral subsets. In this study, we compared the patterns of HIV distribution in cell-free (blood plasma) and cell-associated viruses (peripheral blood mononuclear cells, PBMCs) derived from ART-treated patients by using Sanger sequencing- and Next-Generation sequencing-based HIV assay. CD4+CD45RA−RO+ memory T-cells were isolated from PBMCs using a BD FACSAria instrument. HIV pol (protease and reverse transcriptase) was RT-PCR or PCR amplified from the plasma and the T-cell subset, respectively. Sequences were obtained using Sanger sequencing and Next-Generation Sequencing (NGS). Sanger sequences were aligned and edited using RECall software (beta v3.03). The Stanford HIV database was used to evaluate drug resistance mutations. Illumina MiSeq platform and HyDRA Web were used to generate and analyze NGS data, respectively. Our results show a high correlation between Sanger sequencing and NGS results. However, some major and minor drug resistance mutations were only observed by NGS, albeit at different frequencies. Analysis of low-frequency drugs resistance mutations and virus distribution in the blood compartments may provide information to allow a more sustainable response to therapy and better disease management.

scholarly journals Pulmonary coinfection of Mycobacterium tuberculosis and Tropheryma whipplei: a case report

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Binghua Zhu ◽  
Jing Tang ◽  
Rong Fang ◽  
Xuejie Fei ◽  
Qing Wang ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Mycobacterium Tuberculosis ◽  
Next Generation Sequencing ◽  
Tropheryma Whipplei ◽  
Diagnostic Methods ◽  
Next Generation ◽  
Computed Tomography Scan ◽  
Chest Computed Tomography ◽  
Tomography Scan ◽  
Generation Sequencing

Abstract Background We diagnosed a clinical case of pulmonary infection involving Mycobacterium tuberculosis and Tropheryma whipplei in a patient with acute respiratory distress syndrome. The diagnosis was assisted by metagenomic next-generation sequencing of bronchoalveolar lavage fluid. Case presentation A 44-year-old Han Chinese inmate was transferred to the emergency department because of dry cough, chest tightness, and shortness of breath. The patient’s body temperature rose to 39.3 °C following empirical cephalosporin treatment for 1 week. The blood CD4+/CD8+ ratio was 0.7, suggesting immunodeficiency. Routine microbiological tests were performed, and tuberculosis interferon gamma release assays were positive. Mycobacterium tuberculosis polymerase chain reaction was also positive. Chest computed tomography scan revealed miliary nodules and ground-glass opacifications, which were in accordance with tuberculosis. To fully examine the etiology, we performed routine laboratory tests and metagenomic sequencing, the results of which indicated the presence of Mycobacterium tuberculosis and Tropheryma whipplei. We administered anti-tuberculosis regimen in combination with trimethoprim/sulfamethoxazole. The patient recovered, with chest computed tomography scan showing absorption of lesions. Conclusions Compared with traditional diagnostic methods such as culture and serology, metagenomic next-generation sequencing has the advantage of detecting a wide array of microorganisms in a single test and therefore can be used for clinical diagnosis of rare pathogens and microbial coinfections. It is particularly useful for immunocompromised patients as they are more prone to infection by opportunistic microorganisms.

scholarly journals HIV Drug Resistance Mutations Detection by Next-Generation Sequencing during Antiretroviral Therapy Interruption in China

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 264
Author(s):  
Miaomiao Li ◽  
Shujia Liang ◽  
Chao Zhou ◽  
Min Chen ◽  
Shu Liang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Antiretroviral Therapy ◽  
Detection Threshold ◽  
Next Generation ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Mutations ◽  
Therapy Interruption ◽  
Generation Sequencing

Patients with antiretroviral therapy interruption have a high risk of virological failure when re-initiating antiretroviral therapy (ART), especially those with HIV drug resistance. Next-generation sequencing may provide close scrutiny on their minority drug resistance variant. A cross-sectional study was conducted in patients with ART interruption in five regions in China in 2016. Through Sanger and next-generation sequencing in parallel, HIV drug resistance was genotyped on their plasma samples. Rates of HIV drug resistance were compared by the McNemar tests. In total, 174 patients were included in this study, with a median 12 (interquartile range (IQR), 6–24) months of ART interruption. Most (86.2%) of them had received efavirenz (EFV)/nevirapine (NVP)-based first-line therapy for a median 16 (IQR, 7–26) months before ART interruption. Sixty-one (35.1%) patients had CRF07_BC HIV-1 strains, 58 (33.3%) CRF08_BC and 35 (20.1%) CRF01_AE. Thirty-four (19.5%) of the 174 patients were detected to harbor HIV drug-resistant variants on Sanger sequencing. Thirty-six (20.7%), 37 (21.3%), 42 (24.1%), 79 (45.4%) and 139 (79.9) patients were identified to have HIV drug resistance by next-generation sequencing at 20% (v.s. Sanger, p = 0.317), 10% (v.s. Sanger, p = 0.180), 5% (v.s. Sanger, p = 0.011), 2% (v.s. Sanger, p < 0.001) and 1% (v.s. Sanger, p < 0.001) of detection thresholds, respectively. K65R was the most common minority mutation, of 95.1% (58/61) and 93.1% (54/58) in CRF07_BC and CRF08_BC, respectively, when compared with 5.7% (2/35) in CRF01_AE (p < 0.001). In 49 patients that followed-up a median 10 months later, HIV drug resistance mutations at >20% frequency such as K103N, M184VI and P225H still existed, but with decreased frequencies. The prevalence of HIV drug resistance in ART interruption was higher than 15% in the survey. Next-generation sequencing was able to detect more minority drug resistance variants than Sanger. There was a sharp increase in minority drug resistance variants when the detection threshold was below 5%.

scholarly journals Draft Genome Sequence of Saccharomyces cerevisiae Strain Awamori Number 101, Commonly Used to Make Awamori, a Traditional Spirit, in Okinawa, Japan

2021 ◽  
Vol 10 (25) ◽  
Author(s):  
Masatoshi Tsukahara ◽  
Kotaro Ise ◽  
Maiko Nezuo ◽  
Haruna Azuma ◽  
Takeshi Akao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Next Generation Sequencing ◽  
Genome Sequence ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Next Generation ◽  
Industrial Strain ◽  
Content Type ◽  
Short Reads ◽  
Generation Sequencing

We report here the draft genome sequence for Saccharomyces cerevisiae strain Awamori number 101, an industrial strain used for producing awamori, a distilled alcohol beverage. It was constructed by assembling the short reads obtained by next-generation sequencing. The 315 contigs constitute an 11.5-Mbp genome sequence coding 6,185 predicted proteins.

scholarly journals Identification of Genetic Hereditary Predisposition to Hematologic Malignancies By Clinical Next-Generation Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3854-3854 ◽  
Author(s):  
Amy E Knight Johnson ◽  
Lucia Guidugli ◽  
Kelly Arndt ◽  
Gorka Alkorta-Aranburu ◽  
Viswateja Nelakuditi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Family Members ◽  
Hematologic Malignancies ◽  
Dyskeratosis Congenita ◽  
Molecular Diagnostic ◽  
Next Generation ◽  
Hereditary Predisposition ◽  
Ngs Data ◽  
Generation Sequencing

Abstract Introduction: Myelodysplastic syndrome (MDS) and acute leukemia (AL) are a clinically diverse and genetically heterogeneous group of hematologic malignancies. Familial forms of MDS/AL have been increasingly recognized in recent years, and can occur as a primary event or secondary to genetic syndromes, such as inherited bone marrow failure syndromes (IBMFS). It is critical to confirm a genetic diagnosis in patients with hereditary predisposition to hematologic malignancies in order to provide prognostic information and cancer risk assessment, and to aid in identification of at-risk or affected family members. In addition, a molecular diagnosis can help tailor medical management including informing the selection of family members for allogeneic stem cell transplantation donors. Until recently, clinical testing options for this diverse group of hematologic malignancy predisposition genes were limited to the evaluation of single genes by Sanger sequencing, which is a time consuming and expensive process. To improve the diagnosis of hereditary predisposition to hematologic malignancies, our CLIA-licensed laboratory has recently developed Next-Generation Sequencing (NGS) panel-based testing for these genes. Methods: Thirty six patients with personal and/or family history of aplastic anemia, MDS or AL were referred for clinical diagnostic testing. DNA from the referred patients was obtained from cultured skin fibroblasts or peripheral blood and was utilized for preparing libraries with the SureSelectXT Enrichment System. Libraries were sequenced on an Illumina MiSeq instrument and the NGS data was analyzed with a custom bioinformatic pipeline, targeting a panel of 76 genes associated with IBMFS and/or familial MDS/AL. Results: Pathogenic and highly likely pathogenic variants were identified in 7 out of 36 patients analyzed, providing a positive molecular diagnostic rate of 20%. Overall, 6 out of the 7 pathogenic changes identified were novel. In 2 unrelated patients with MDS, heterozygous pathogenic sequence changes were identified in the GATA2 gene. Heterozygous pathogenic changes in the following autosomal dominant genes were each identified in a single patient: RPS26 (Diamond-Blackfan anemia 10), RUNX1 (familial platelet disorder with propensity to myeloid malignancy), TERT (dyskeratosis congenita 4) and TINF2 (dyskeratosis congenita 3). In addition, one novel heterozygous sequence change (c.826+5_826+9del, p.?) in the Fanconi anemia associated gene FANCA was identified. . The RNA analysis demonstrated this variant causes skipping of exon 9 and results in a premature stop codon in exon 10. Further review of the NGS data provided evidence of an additional large heterozygous multi-exon deletion in FANCA in the same patient. This large deletion was confirmed using array-CGH (comparative genomic hybridization). Conclusions: This study demonstrates the effectiveness of using NGS technology to identify patients with a hereditary predisposition to hematologic malignancies. As many of the genes associated with hereditary predisposition to hematologic malignancies have similar or overlapping clinical presentations, analysis of a diverse panel of genes is an efficient and cost-effective approach to molecular diagnostics for these disorders. Unlike Sanger sequencing, NGS technology also has the potential to identify large exonic deletions and duplications. In addition, RNA splicing assay has proven to be helpful in clarifying the pathogenicity of variants suspected to affect splicing. This approach will also allow for identification of a molecular defect in patients who may have atypical presentation of disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assay of frequency and spectrum of genetic variants in TTN in healthy russian population

2021 ◽  
Vol 8 (5) ◽  
pp. 29-37
Author(s):  
Yu. A. Vakhrushev ◽  
A. A. Kozyreva ◽  
S. V. Zhuk ◽  
O. P. Rotar ◽  
A. A. Kostareva
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Sanger Sequencing ◽  
Heart Diseases ◽  
Genetic Research ◽  
Russian Population ◽  
Next Generation ◽  
Data Bases ◽  
International Data ◽  
Generation Sequencing

Background. Gene TTN associated with all types of cardiomyopathy, however its large size (294 b.p.) warrants a lot of individual unique genetic variants or variants with low frequency, that aggravates their interpretation. Besides that nowadays there is no data about spectrum of variants in this gene in healthy Russian population. Recognition frequency and spectrum of variants in gene TTN in healthy Russian population will allow us to use it for interpretation results of molecular genetic research for patients with different heart pathology, and define prognosis for different heart diseases.Objective. Recognize frequency and spectrum of single nucleotide and truncating variants in gene TTN in healthy Russian population and compare it with international data bases, and evaluate level of pathogenicity these variants and their distributing across titin structure.Design and methods. 192 men in age 55,8±6,6 years were tested with next-generation sequencing. Identified genetic variants were confirmed by Sanger sequencing. Results. Allele missense variant frequency (with frequency less than 0.1%) in TTN in healthy Russian population amount to 15.1 %, and truncating variants — 0.52 %. 37,9 % of them were variants of unknown significance, 62 % — likely-benign and 0.1 % — benign. There was no pathological and likely-pathological variants. Identified genetic variants distributed throughout the titin structure.Conclusion. Received result is congruent с international data bases and researches. Expended laboratory method (Next generation sequencing and confirmation with Sanger sequencing) can be used both in clinical practice, and in creating data bases of genetic variants in healthy Russian population.

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