Multicenter validation study for the certification of a CFTR gene scanning method using next generation sequencing technology

2018 ◽  
Vol 56 (7) ◽  
pp. 1046-1053 ◽  
Author(s):  
Anne Bergougnoux ◽  
Valeria D’Argenio ◽  
Stefanie Sollfrank ◽  
Fanny Verneau ◽  
Antonella Telese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Genetic Analysis ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Sequencing Data ◽  
Scanning Method ◽  
Next Generation Sequencing Technology ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom’s CFTR MASTR Dx kit to obtain CE-IVD certification. Methods: A total of 164 samples, previously analyzed with well-established “reference” methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data. Results and conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

scholarly journals cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

2021 ◽  
Author(s):  
Jasmina Damnjanović ◽  
Nana Odake ◽  
Jicheng Fan ◽  
Beixi Jia ◽  
Takaaki Kojima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Substrate Preference ◽  
Stable Complex ◽  
Next Generation ◽  
Library Size ◽  
Binary Model ◽  
Wide Range ◽  
Generation Sequencing ◽  
Selection Of

AbstractcDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

scholarly journals ECCsplorer: a pipeline to detect extrachromosomal circular DNA (eccDNA) from next-generation sequencing data

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Ludwig Mann ◽  
Kathrin M. Seibt ◽  
Beatrice Weber ◽  
Tony Heitkam
Keyword(s):  
Next Generation Sequencing ◽  
Transposable Elements ◽  
Data Availability ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Circular Dna ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background Extrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. Apart from carrying tandemly repeated DNA, eccDNAs may also harbor extra copies of genes or recently activated transposable elements. As eccDNAs occur in all eukaryotes investigated so far and likely play roles in stress, cancer, and aging, they have been prime targets in recent research—with their investigation limited by the scarcity of computational tools. Results Here, we present the ECCsplorer, a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing techniques. Following Illumina-sequencing of amplified circular DNA (circSeq), the ECCsplorer enables an easy and automated discovery of eccDNA candidates. The data analysis encompasses two major procedures: first, read mapping to the reference genome allows the detection of informative read distributions including high coverage, discordant mapping, and split reads. Second, reference-free comparison of read clusters from amplified eccDNA against control sample data reveals specifically enriched DNA circles. Both software parts can be run separately or jointly, depending on the individual aim or data availability. To illustrate the wide applicability of our approach, we analyzed semi-artificial and published circSeq data from the model organisms Homo sapiens and Arabidopsis thaliana, and generated circSeq reads from the non-model crop plant Beta vulgaris. We clearly identified eccDNA candidates from all datasets, with and without reference genomes. The ECCsplorer pipeline specifically detected mitochondrial mini-circles and retrotransposon activation, showcasing the ECCsplorer’s sensitivity and specificity. Conclusion The ECCsplorer (available online at https://github.com/crimBubble/ECCsplorer) is a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing data. The derived eccDNA targets are valuable for a wide range of downstream investigations—from analysis of cancer-related eccDNAs over organelle genomics to identification of active transposable elements.

scholarly journals Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255257
Author(s):  
Daichi Sadato ◽  
Chizuko Hirama ◽  
Ai Kaiho-Soma ◽  
Ayaka Yamaguchi ◽  
Hiroko Kogure ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Molecular Diagnosis ◽  
Driver Gene ◽  
Next Generation ◽  
Sequencing Data ◽  
Dna And Rna ◽  
Myeloid Neoplasms ◽  
Wide Range ◽  
Generation Sequencing

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

scholarly journals Development of the TypeSeq Assay for Detection of 51 Human Papillomavirus Genotypes by Next-Generation Sequencing

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Sarah Wagner ◽  
David Roberson ◽  
Joseph Boland ◽  
Meredith Yeager ◽  
Michael Cullen ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Next Generation Sequencing ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Perfect Agreement ◽  
Hpv Genotyping ◽  
Wide Range ◽  
Positive Agreement ◽  
Generation Sequencing ◽  
Multiple Type

ABSTRACTWe have developed a new human papillomavirus (HPV) genotyping assay for detection of 51 HPV genotypes by next-generation sequencing (NGS). The TypeSeq assay consists of 3 PCR steps that equalize viral load and each type’s amplicon copies prior to genotyping by NGS, thereby maximizing multiple-type sensitivity with minimal sequencing reads. The analytical sensitivity of the TypeSeq assay is 10 copies per reaction for 49 of the 51 types, including 13 high-risk (HR) types. We tested 863 clinical cervical specimens previously evaluated with the Roche Linear Array HPV genotyping test (LA). TypeSeq achieved 94.4% positive agreement with LA for detection of any HR type. Positive agreement was 91.4% and 85.5% for HPV16 and HPV18, respectively. Low-risk (LR) types ranged from 40.0% positive agreement (HPV83) to 90.9% (HPV69). Our unique approach to HPV amplification achieved a multiple-type sensitivity comparable to that of LA, with 83.9% and 84.2% of specimens positive for multiple HPV types by TypeSeq or LA, respectively. A total of 48.2% of specimens showed perfect agreement for all 37 types common to both assays. The simplicity of our open-source TypeSeq assay allows for high-throughput yet scalable processing, with a single technician able to process up to 768 specimens within 3 days. By leveraging NGS sample multiplexing capabilities, the per-sample labor requirements are greatly reduced compared to those of traditional genotyping methods. These features and the broad spectrum of detectable types make TypeSeq highly suitable for a wide range of applications.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown. Methods Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1. Results The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44. Conclusion In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background: Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown.Methods: Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1.Results: The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44.Conclusion: In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals Genotyping of 25 Leukemia-Associated Genes in a Single Work Flow by Next-Generation Sequencing Technology with Low Amounts of Input Template DNA

2013 ◽  
Vol 59 (8) ◽  
pp. 1238-1250 ◽  
Author(s):  
Jenny Rinke ◽  
Vivien Schäfer ◽  
Mathias Schmidt ◽  
Janine Ziermann ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Target Genes ◽  
Work Flow ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Wide Range ◽  
Next Generation Sequencing Ngs ◽  
Template Dna ◽  
Ngs Data ◽  
Generation Sequencing

BACKGROUND We sought to establish a convenient, sensitive next-generation sequencing (NGS) method for genotyping the 26 most commonly mutated leukemia-associated genes in a single work flow and to optimize this method for low amounts of input template DNA. METHODS We designed 184 PCR amplicons that cover all of the candidate genes. NGS was performed with genomic DNA (gDNA) from a cohort of 10 individuals with chronic myelomonocytic leukemia. The results were compared with NGS data obtained from sequencing of DNA generated by whole-genome amplification (WGA) of 20 ng template gDNA. Differences between gDNA and WGA samples in variant frequencies were determined for 2 different WGA kits. RESULTS For gDNA samples, 25 of 26 genes were successfully sequenced with a sensitivity of 5%, which was achieved by a median coverage of 492 reads (range, 308–636 reads) per amplicon. We identified 24 distinct mutations in 11 genes. With WGA samples, we reliably detected all mutations above 5% sensitivity with a median coverage of 506 reads (range, 256–653 reads) per amplicon. With all variants included in the analysis, WGA amplification by the 2 kits tested yielded differences in variant frequencies that ranged from −28.19% to +9.94% [mean (SD) difference, −0.2% (4.08%)] and from −35.03% to +18.67% [mean difference, −0.75% (5.12%)]. CONCLUSIONS Our method permits simultaneous analysis of a wide range of leukemia-associated target genes in a single sequencing run. NGS can be performed after WGA of template DNA for reliable detection of variants without introducing appreciable bias.

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