scholarly journals Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rajib Majumder ◽  
Brodie Sutcliffe ◽  
Phillip W. Taylor ◽  
Toni A. Chapman
Keyword(s):  
Next Generation Sequencing ◽  
Illumina Miseq ◽  
Fruit Fly ◽  
Egg Laying ◽  
Food Sources ◽  
Natural Food ◽  
Next Generation ◽  
North East ◽  
Host Fruit ◽  
Generation Sequencing

Abstract Insects typically host substantial microbial communities (the ‘microbiome’) that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni (‘Queensland fruit fly’) that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect’s microbiome and that of the surrounding host tissues.

Targeted next-generation sequencing as a diagnostic tool in gastrointestinal system cancer/polyposis patients

2020 ◽  
Vol 106 (6) ◽  
pp. 510-517
Author(s):  
Sinem Yalcintepe ◽  
Hakan Gurkan ◽  
Selma Demir ◽  
Hilmi Tozkir ◽  
Huseyin Ahmet Tezel ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gastrointestinal Cancer ◽  
Illumina Miseq ◽  
Cancer Predisposition ◽  
Next Generation ◽  
Personalized Care ◽  
Screening Programs ◽  
Predisposition Genes ◽  
Generation Sequencing ◽  
Dependent Probe

Background: Recent advances in next-generation sequencing (NGS) technology have enabled multigene testing and changed the diagnostic approach to hereditary gastrointestinal cancer/polyposis syndromes. The aim of this study was to analyze different cancer predisposition genes in hereditary/sporadic gastrointestinal cancer/polyposis. Methods: Cancer predisposition genes were analyzed with an Illumina MiSeq NGS system in 80 patients with gastrointestinal cancer/polyposis who were examined between the years 2016 and 2019. Deletion/duplication analysis of MLH1, MSH2, and EPCAM genes was performed by using the multiplex ligation-dependent probe amplification method. Results: Germline testing of hereditary cancer-related genes was performed in 80 patients with gastrointestinal cancer/polyposis. A total of 30 variants in 30 cases (37.5%) were assessed as pathogenic/likely pathogenic. A total of 19 heterozygous variants were assessed as variants of uncertain clinical significance in 17 cases (21.25%) and 18 (22.5%) novel variations (9 pathogenic/likely pathogenic, 9 variants of uncertain significance) were determined. In 4 (5%) cases, multiplex ligation-dependent probe amplification detected deletions in MLH1, MSH2, and EPCAM genes. Conclusion: The accumulation of analyses with multigene testing will increase the available data for cancer predisposition genes in hereditary gastrointestinal cancer/polyposis. Educational campaigns for prevention, efficient screening programs, and more personalized care based on the profile of individual patients are necessary.

scholarly journals Pengembangan Metode In-House HLA-Typing Gen HLA Kelas I (HLA A, HLA B, dan HLA C) Menggunakan Next Generation Sequencing Illumina MiSeq

2015 ◽  
Vol 47 (3) ◽  
pp. 152-159
Author(s):  
Rika Yuliwulandari ◽  
Kinasih Prayuni ◽  
Kenconoviyati ◽  
R. W. Susilowati ◽  
Abdul Salam M. Sofro
Keyword(s):  
Next Generation Sequencing ◽  
Illumina Miseq ◽  
Hla Typing ◽  
Next Generation ◽  
Generation Sequencing

Moving next-generation sequencing into the clinical realm: Detection of somatic mutations in cancer by targeted amplicon sequencing.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 60-60
Author(s):  
Chih-Jian Lih ◽  
Thomas Forbes ◽  
Michele Mehaffey ◽  
Eric Sause ◽  
David Sims ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Next Generation Sequencing ◽  
Cancer Patients ◽  
Patient Selection ◽  
Somatic Mutations ◽  
Germ Line ◽  
Early Stage ◽  
Illumina Miseq ◽  
Next Generation ◽  
Generation Sequencing

60 Background: Molecular targeted therapies are increasingly important in treating cancer patients; robust analytically validated clinical assays are required for patient selection in early-stage clinical trials. The goal of Molecular Characterization Laboratory (MoCha) is to develop clinical diagnostic assays using next generation sequencing methods to support clinical studies in DCTD (CTEP). Methods: We developed a custom assay for somatic mutation detection using Fluidigm access array technology for amplicon generation followed by sequencing with the Illumina Miseq. A panel of 62 amplicons covering 6 Kb genomic regions was designed to detect 92 DNA loci, including common therapeutically actionable targets, in 37 genes. Analytical studies were performed using genomic DNA samples from fresh or formalin fixed cancer cell-lines and a normal hapmap individual (CEPH). We subsequently applied this assay to characterize DNA samples from both tumor tissues and blood specimens from ovarian cancer patients. Results: The assay detected known variants in both frozen and fixed DNA samples reproducibly with high sensitivity and specificity (<2%). Using a series of positive control plasmid spikes mixed into a normal reference CEPH DNA at pre-defined copy number ratios, we verified the assay is sensitive to detect variants at 5% allelic frequency with a minimum 400 X coverage. We identified somatic mutations in TP53 and PIK3CA in a few patients, and a germ-line variant D1583N in ATM genes occurring in one-third of tested patients. Conclusions: We developed and validated a next generation sequencing assay suitable for patient selection for clinical trials. Plans are to correlate sequencing and clinical results when clinical data are available.

scholarly journals Microbiota in Waterlogged Archaeological Wood: Use of Next-Generation Sequencing to Evaluate the Risk of Biodegradation

2020 ◽  
Vol 10 (13) ◽  
pp. 4636 ◽  
Author(s):  
Federica Antonelli ◽  
Alfonso Esposito ◽  
Giulia Galotta ◽  
Barbara Davidde Petriaggi ◽  
Silvano Piazza ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Background Information ◽  
Archaeological Sites ◽  
Next Generation ◽  
Archaeological Wood ◽  
Waterlogged Archaeological Wood ◽  
Sequencing Chemistry ◽  
Generation Sequencing

Waterlogged archaeological wood (WAW) is considered a precious material, first-hand account of past civilizations. Like any organic material, it is subjected to biodegradative action of microorganisms whose activity could be particularly fast and dangerous during the phases of excavation, storage and restoration. The present work aimed to characterize the microorganisms present in WAW during these tricky periods to evaluate the biological risk it is exposed to. The bacterial and fungal communities inhabiting woods coming from two archaeological sites (Pisa and Naples) were investigated through Next-Generation Sequencing (NGS). High-throughput sequencing of extracted DNA fragments was performed using the reversible terminator-based sequencing chemistry with the Illumina MiSeq platform. The analyses revealed that the two archaeological sites showed distinct richness and biodiversity, as expected. In all the WAWs, the bacterial community harbored mainly Proteobacteria, whereas Bacteroidetes was well represented only in Naples communities and taxa belonging to the phyla Chloroflexi only in the Pisa site. Concerning the fungal community, the two sites were dominated by different phyla: Ascomycota for Naples samples and Basidiomycota for Pisa. Interestingly, most of the identified bacterial and fungal taxa have cellulolytic or ligninolytic ability. These results provide new and useful background information concerning the composition of WAW microbiota and the threat it represents for this precious material.

scholarly journals The study of hybridization processes within genus Sparganium L. Subgenus Xanthosparganium holmb. Based on data of next generation sequencing (NGS)

2019 ◽  
Vol 17 (4) ◽  
pp. 27-35
Author(s):  
Evgeniy A. Belyakov ◽  
Eduard M. Machs ◽  
Yulia V. Mikhailova ◽  
Aleksandr V. Rodionov
Keyword(s):  
Next Generation Sequencing ◽  
North American ◽  
Illumina Miseq ◽  
Rrna Genes ◽  
Next Generation ◽  
Close Relationship ◽  
Species Characteristics ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The study represents the results of research of intragenic polymorphism in transcribed spacer ITS1 of the 35S rRNA genes in representatives of subgenus Xanthosparganium genus Sparganium which were obtained by means of locus-specific next generation sequencing on the platform Illumina MiSeq. It was shown that ribotype variations in studied samples generally correspond to the division of this genus into three sections Erecta (subgenus Sparganium), Natantia and Minima (subgenus Xanthosparganium). High level of intragenic polymorphism was revealed in S. hyperboreum, with ribotypes distributed among several groups. Genome of this species includes ribotypes which are typical for other species in subgenus Xanthosparganium. For two investigated S. glomeratum samples, there were no ribotypes similar to such ribotypes in other species of Natantia section. S. glomeratum has got ribotypes identical with S. hyperboreum of Minima section. This feature may be the evidence of ancient intersectional hybridization of these two species. Characteristics of rDNA in S. glomeratum are in favor of putting this species into Minima section. It was suggested that speciation processes within the genus could be based not only on hybridization but also went on in allopatric way. The fist statement is supported by the presence of similar and identical ribotypes in S. emersum, S. longifolium, S. gramineum and S. hyperboreum, the second as it was mentioned by other researchers, is due to close relationship between North American and Eurasian taxa.

scholarly journals Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearranged mitochondrial genomes inAmolops chunganensisandQuasipaa boulengeri

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2786 ◽  
Author(s):  
Siqi Yuan ◽  
Yun Xia ◽  
Yuchi Zheng ◽  
Xiaomao Zeng
Keyword(s):  
Next Generation Sequencing ◽  
Gene Order ◽  
Illumina Miseq ◽  
Mitochondrial Genomes ◽  
Trna Genes ◽  
Gene Rearrangements ◽  
Next Generation ◽  
Protein Coding ◽  
Alignment Errors ◽  
Generation Sequencing

Recent improvements in next-generation sequencing (NGS) technologies can facilitate the obtainment of mitochondrial genomes. However, it is not clear whether NGS could be effectively used to reconstruct the mitogenome with high gene rearrangement. These high rearrangements would cause amplification failure, and/or assembly and alignment errors. Here, we choose two frogs with rearranged gene order,Amolops chunganensisandQuasipaa boulengeri, to test whether gene rearrangements affect the mitogenome assembly and alignment by using NGS. The mitogenomes with gene rearrangements are sequenced through Illumina MiSeq genomic sequencing and assembled effectively by Trinity v2.1.0 and SOAPdenovo2. Gene order and contents in the mitogenome ofA. chunganensisandQ. boulengeriare typical neobatrachian pattern except for rearrangements at the position of “WANCY” tRNA genes cluster. Further, the mitogenome ofQ. boulengeriis characterized with a tandem duplication oftrnM. Moreover, we utilize 13 protein-coding genes ofA. chunganensis,Q. boulengeriand other neobatrachians to reconstruct the phylogenetic tree for evaluating mitochondrial sequence authenticity ofA. chunganensisandQ. boulengeri. In this work, we provide nearly complete mitochondrial genomes ofA. chunganensisandQ. boulengeri.

scholarly journals Development and Dynamics of Cytomegalovirus UL97 Ganciclovir Resistance Mutations in Transplant Recipients Detected by Next Generation Sequencing

2021 ◽  
Author(s):  
Isabelle P Lodding ◽  
Mette Jørgensen ◽  
Marc Bennedbæk ◽  
Nikolai Kirkby ◽  
Klaudia Naegele ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Illumina Miseq ◽  
Primary Prophylaxis ◽  
Solid Organ ◽  
Next Generation ◽  
Induced Mutations ◽  
Resistance Mutations ◽  
Hematopoietic Stem ◽  
Ganciclovir Resistance ◽  
Generation Sequencing

Abstract Background (Val)ganciclovir resistance mutations in CMV UL97 (UL97-GCV-R) complicate anti-CMV therapy in recipients of solid organ and hematopoietic stem-cell transplants but comprehensive data on prevalence, emergence, and outcome are scarce. Methods Using next generation sequencing (NGS) (Illumina MiSeq platform), we analysed UL97-GCV-R in patients with available plasma samples and refractory CMV replication/DNAemia (n=87) containing viral loads ≥910 IU/mL. 21 patients with CMV DNAemia resolving under antiviral therapy were analysed as controls. Detected mutations were considered induced and of potential clinical significance if they increased by ≥10% compared to the first detected frequency, or if they had a maximum frequency ≥25%. Results 19/87 (21.8%) with refractory CMV replication had &gt;1 UL97-GCV-R detected by NGS, in comparison to 0/21 of the controls (p=0.02). One third of the recipients had 2 or more induced UL97-GCV-R mutations. The most frequently induced mutations affected codons 595 (42% (8/19)), 594 (32% (6/19)) and 603 (32% (6/19)). C592G was present in all episodes of both cases and controls at frequencies &lt;15%, but never induced. UL97-GCV-R tended to be more frequent in donor/recipient CMV IgG mismatch or following failure to complete primary prophylaxis, and many developed invasive CMV disease. Conclusion UL97-GCV-R is common among transplant patients with refractory CMV replication. Early testing by NGS allows for identification of major mutations at codons 595, 594 and 603, and exclude a major role of C592G in ganciclovir resistance. Large prospective studies on UL97-GCV-R are warranted.

scholarly journals Detection of a Single-Copy Plasmid, pXFSL21, in Xylella fastidiosa Strain Stag’s Leap with Two Toxin-Antitoxin Systems Using Next-Generation Sequencing

2019 ◽  
Vol 109 (2) ◽  
pp. 240-247 ◽  
Author(s):  
Christopher Van Horn ◽  
Fengnian Wu ◽  
Zheng Zheng ◽  
Zehan Dai ◽  
Jianchi Chen
Keyword(s):  
Next Generation Sequencing ◽  
Xylella Fastidiosa ◽  
Bacterial Genome ◽  
Illumina Miseq ◽  
Single Copy ◽  
Next Generation ◽  
In Planta ◽  
Term Survival ◽  
Generation Sequencing

Plasmids are important genetic elements contributing to bacterial evolution and environmental adaptation. Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing economically devastating diseases such as Pierce’s disease (PD) of grapevine. In this study, the plasmid status of a highly virulent PD strain, Stag’s Leap, originally isolated from Napa Valley, CA, was studied using sequencing and bioinformatics tools. DNA samples extracted from a pure culture in periwinkle wilt medium (in vitro DNA) and a PD-symptomatic grapevine artificially inoculated in the greenhouse (in planta DNA) were subject to next-generation sequencing (NGS) analyses (Illumina MiSeq or HiSeq). Sequence analyses and polymerase chain reaction experiments revealed the presence of a circular plasmid, pXFSL21, of 21,665 bp. This plasmid existed as a single copy per bacterial genome under both in vitro and in planta conditions. Two toxin-antitoxin (T-A) systems (ydcD-ydcE and higB-higA) were detected in pXFSL21, a possible mechanism for the long-term survival of this single-copy plasmid in the bacterial population. BLAST searches against the GenBank database (version 222) detected homologs of the two T-A systems in chromosomes or plasmids of some X. fastidiosa strains. However, double T-A systems were found only in pXFSL21. pXFSL21 was not found in other known PD strains and, therefore, could serve as a molecular marker for strain Stag’s Leap monitoring and tracking. The NGS-based technique outlined in this article provides an effective tool for identifying single- or low-copy-number plasmids in fastidious prokaryotes.

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.

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