Combining LC-MS metabolomics and next generation sequencing to study the interactions between herbal medicines and human gut bacteria in-vitro

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.

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Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

Journal of Virology ◽

10.1128/jvi.01900-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 2

Author(s):

Marilia Rita Pinzone ◽

Maria Paola Bertuccio ◽

D. Jake VanBelzen ◽

Ryan Zurakowski ◽

Una O’Doherty

Keyword(s):

T Cells ◽

Next Generation Sequencing ◽

Cd4 T Cells ◽

Next Generation ◽

Activated T Cells ◽

Selection Pressures ◽

Hiv Dna ◽

Generation Sequencing

ABSTRACT Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo. When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

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Genome study of PDGFRA D842V mutant GIST using next generation sequencing approach.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.10540 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 10540-10540

Author(s):

Maria A. Pantaleo ◽

Annalisa Astolfi ◽

Milena Urbini ◽

Valentina Indio ◽

Margherita Nannini ◽

...

Keyword(s):

Next Generation Sequencing ◽

Tyrosine Kinases ◽

Therapeutic Targets ◽

Mapk Signaling ◽

Gastric Gist ◽

Next Generation ◽

Single Nucleotide Variants ◽

Generation Sequencing

10540 Background: Mutations of the receptors KIT and PDGFRA in GIST are the oncogenetic events of disease as well as the targets of molecular therapies. Within the PDGFRA mutations, the D842V mutation in exon 18 confers in vitro and in vivo resistance to imatinib. Next generation sequencing techniques may completely dissect all the possible somatic mutations and genomic rearrangements in order to identify novel therapeutic targets in this patients population. Methods: Five patients with gastric GIST were analyased (3 M, 2 F; mean age 65,5 years, range 51-77). The tumor dimension ranged between 3 and 15 cm, MI < 2 and 8 /50 HPF. No metastases were present in all cases. The KIT and PDGFRA anlysis showed a D842V mutation in exon 18 of PDGFRA in all cases. Whole transcriptome sequencing was performed with Illumina HiScanSQ platform with a paired-end strategy (75x2). After performing quality controls, the short reads were mapped with Tophat-Botie pipeline against the human reference genome (HG19). The variations, such as Single nucleotide variants (SNVs) and InDels, were called by SNVMix2 (a software suited for SNVs discovery in tumor samples) implementing an accurate filtering procedures developed in our laboratory. Two predictors of mutations effect at protein level (SNPs&GO and Provean) were employed in order to prioritize the emerging variation. Results: An average of 206 coding non-synonymous variants were highlighted in the five GIST samples, of which ~ 30% were predicted as deleterious with at least one predictor. In addition to PDGFRA D842V mutation, in all five patients were found mutations on different receptor tyrosine kinases, such as FGFR4 and DDR2. Moreover three out of five patients harboured mutations on members of the MDR/TAP subfamily that are involved in multidrug resistance, in particular on ABCB1, ABCB4 and on ABCB6 genes. Other mutations were found on the hedgehog and MAPK signaling pathway and on SNF/SWI chromatin remodeling complex. Conclusions: New molecular events have been identified in PDGFRA D842V mutant GIST. These data should be validated in larger series and the role of these mutations as therapeutic targets should be further investigated.

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Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

Genes ◽

10.3390/genes5041115 ◽

2014 ◽

Vol 5 (4) ◽

pp. 1115-1131 ◽

Cited By ~ 13

Author(s):

Yukio Kurihara ◽

Yuko Makita ◽

Mika Kawashima ◽

Hidefumi Hamasaki ◽

Yoshiharu Yamamoto ◽

...

Keyword(s):

Transcription Factor ◽

Next Generation Sequencing ◽

Genomic Dna ◽

Dna Fragments ◽

Next Generation ◽

Regulatory Potential ◽

Generation Sequencing

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

10.1101/2021.09.08.459404 ◽

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.

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Identification of potential genes in upper tract urothelial carcinoma using next-generation sequencing with bioinformatics and in vitro analyses

PeerJ ◽

10.7717/peerj.11343 ◽

2021 ◽

Vol 9 ◽

pp. e11343

Author(s):

Hsiang-Ying Lee ◽

Ching-Chia Li ◽

Wei-Ming Li ◽

Ya-Ling Hsu ◽

Hsin-Chih Yeh ◽

...

Keyword(s):

Gene Expression ◽

Next Generation Sequencing ◽

Candidate Genes ◽

Venn Diagram ◽

Next Generation ◽

Upper Tract ◽

Urothelial Carcinomas ◽

Gene And Protein Expression ◽

Generation Sequencing

Background We aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases. Materials and Methods Using next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity® Pathway Analysis databases, we identified candidate genes. To search for upstream miRNAs with exact target binding sites, we used miRmap, TargetScan, and miRDB to enforce evidence. Then, we clarified gene and protein expression through an in vitro study using western blot analysis and quantitative real-time reverse transcriptase-PCR. Results Interactions between selected target genes obtained using the NGS and miRmap methods were assessed through a Venn diagram analysis. Six potential genes, namely, PDE5A, RECK, ZEB2, NCALD, PLCXD3 and CYBRD1 showed significant differences. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC. In addition, we compared the expression of these genes in various UC cell lines (RT4, BFTC905, J82, T24, UMUC3, 5637, BFTC 909, UMUC14) and found decreased expression of PDE5A in muscle-invasive UC cells compared with the RT4 cell line. Furthermore, by using paired UTUC and normal tissues from 20 patients, lower PDE5A expression was also demonstrated in tumor specimens. Conclusions Our findings suggest these candidate genes may play some roles in UTUC progression. We propose that these markers may be potential targets clarified by in vitro and in vivo experiments. PDE5A also potentially presents tumor suppressor genes, as identified by comparing the expression between normal and tumor specimens.

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Insertion Specificity of the hATx-6 Transposase of Hydra magnipapillata

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.734154 ◽

2021 ◽

Vol 8 ◽

Author(s):

Paul Riggs ◽

George Blundell-Hunter ◽

Joanna Hagelberger ◽

Guoping Ren ◽

Laurence Ettwiller ◽

...

Keyword(s):

Next Generation Sequencing ◽

Bioinformatic Analysis ◽

Next Generation ◽

Hydra Vulgaris ◽

Domains Of Life ◽

Hydra Magnipapillata ◽

Sequence Bias ◽

Generation Sequencing

Transposable elements (TE) are mobile genetic elements, present in all domains of life. They commonly encode a single transposase enzyme, that performs the excision and reintegration reactions, and these enzymes have been used in mutagenesis and creation of next-generation sequencing libraries. All transposases have some bias in the DNA sequence they bind to when reintegrating the TE DNA. We sought to identify a transposase that showed minimal sequence bias and could be produced recombinantly, using information from the literature and a novel bioinformatic analysis, resulting in the selection of the hATx-6 transposase from Hydra vulgaris (aka Hydra magnipapillata) for further study. This transposase was tested and shown to be active both in vitro and in vivo, and we were able to demonstrate very low sequence bias in its integration preference. This transposase could be an excellent candidate for use in biotechnology, such as the creation of next-generation sequencing libraries.

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