Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Doctoral Thesis

Turnaround Time ◽

Storage Conditions ◽

Tissue Preparation ◽

Next Generation ◽

Human Donor ◽

Taxonomic Profiling ◽

Storage Media ◽

Hypothermic Storage ◽

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Summary of the Doctoral Thesis

10.25143/prom-rsu_2021-21_dts ◽

2021 ◽

Author(s):

◽

Davide Borroni ◽

Keyword(s):

Turnaround Time ◽

Storage Conditions ◽

Tissue Preparation ◽

Next Generation ◽

Human Donor ◽

Taxonomic Profiling ◽

Storage Media ◽

Hypothermic Storage ◽

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium

BMJ Open Ophthalmology ◽

10.1136/bmjophth-2018-000246 ◽

2019 ◽

Vol 4 (1) ◽

pp. e000246 ◽

Cited By ~ 2

Author(s):

Mohit Parekh ◽

Davide Borroni ◽

Vito Romano ◽

Stephen B Kaye ◽

Davide Camposampiero ◽

...

Keyword(s):

Storage Conditions ◽

Next Generation ◽

Human Donor ◽

Taxonomic Profiling ◽

Storage Media ◽

Hypothermic Storage ◽

Corneal Preservation ◽

And Control ◽

ObjectiveTo detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method.MethodsSeven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments.ResultsIn both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples.ConclusionMetagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Different Methods in HPV Genotyping of Anogenital and Oropharyngeal Lesions: Comparison between VisionArray® Technology, Next Generation Sequencing, and Hybrid Capture Assay

Journal of Molecular Pathology ◽

10.3390/jmp2010004 ◽

2021 ◽

Vol 2 (1) ◽

pp. 29-41

Author(s):

Giorgia Acquaviva ◽

Michela Visani ◽

Viviana Sanza ◽

Antonio De Leo ◽

Thais Maloberti ◽

...

Keyword(s):

Turnaround Time ◽

Hpv Infection ◽

Human Papillomaviruses ◽

Next Generation ◽

Hybrid Capture ◽

Capture Assay ◽

Anogenital Area ◽

Next Generation Sequencing Ngs ◽

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results.

Tumor BRCA Test for Patients with Epithelial Ovarian Cancer: The Role of Molecular Pathology in the Era of PARP Inhibitor Therapy

Cancers ◽

10.3390/cancers11111641 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1641 ◽

Cited By ~ 5

Author(s):

Caterina Fumagalli ◽

Federica Tomao ◽

Ilaria Betella ◽

Alessandra Rappa ◽

Mariarosaria Calvello ◽

...

Keyword(s):

Ovarian Cancer ◽

Epithelial Ovarian Cancer ◽

Success Rate ◽

Parp Inhibitor ◽

Turnaround Time ◽

Molecular Diagnostic ◽

Next Generation ◽

Clinical Implementation ◽

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

Tissue Preparation and Next-generation Sequencing Workflows for Oncology Testing

The Journal of OncoPathology ◽

10.13032/tjop.2052-5931.100051 ◽

2014 ◽

Vol 1 (4) ◽

pp. 115-120

Author(s):

Ronald van Eijk ◽

Stijn Crobach

Keyword(s):

Tissue Preparation ◽

Next Generation ◽

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

10.21203/rs.2.22699/v1 ◽

2020 ◽

Author(s):

Ome Kalsoom Afridi ◽

Johar Ali ◽

Jeong Ho Chang

Keyword(s):

Microbial Communities ◽

Broiler Chickens ◽

Human Subjects ◽

Fecal Microbiota ◽

Next Generation ◽

Microbial Association ◽

Taxonomic Profiling ◽

Microbial Dysbiosis ◽

Abstract Background: Microbiota plays an important role in food safety and its alteration poses a serious threat to humans. Comparative microbiome profiling using next-generation sequencing (NGS) enabled the understanding of microbial diversity and similarity between different species. In this study, we used NGS to profile the fecal microbiota of sick human and broiler chickens. A total of 26 fecal samples were collected from severely sick human subjects (n= 13) and broiler chickens (n=13) with similar symptoms. Results: The total number of microbial species detected in broiler chickens fecal microbiota was higher than that of humans. Phylum Proteobacteria was the most abundant in both human and broiler chickens fecal microbiota while Tenericutes was found to be least abundant in both species. Phylum Actinobacteria was found only in the human fecal microbiota. In both humans and broiler chickens, E.coli was found to be phylogenetically related suggesting a microbial association between both species. Conclusion: NGS based taxonomic profiling revealed the association of microbial dysbiosis with extreme sickness in both humans and broiler chickens. The dominance of phylum Proteobacteria in both the species ascertains their altered gut microbiota. Both human and broiler chickens microbial communities were found to be genetically related indicating horizontal transfer of microbes between the two species.

Two-Year Experience of Performing a Next-Generation-Sequencing Based Panel Test in an Academic Medical Center and Its Clinical Impact

Blood ◽

10.1182/blood.v128.22.1707.1707 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1707-1707 ◽

Cited By ~ 1

Author(s):

Kuo Frank ◽

Jon C. Aster ◽

Coleman Lindsley

Keyword(s):

Academic Medical Center ◽

Prognostic Significance ◽

Turnaround Time ◽

Genetic Alterations ◽

Splenic Marginal Zone Lymphoma ◽

Next Generation ◽

Lymphoid Malignancies ◽

Myeloid Neoplasm ◽

Abstract Our ability to interrogate a broad array of genetic alterations in myeloid neoplasm has increased significantly with the advance in next-generation sequencing (NGS). In addition to morphologic examination, flow cytometry and cytogenetics, NGS-based testing can add additional useful information to the diagnostic workup. With improved turnaround time, decreasing costs and an expanding knowledge of the therapeutic and prognostic significance of the detected variants, NGS-based panel testing has increasingly played a major role in the management of patients with myeloid neoplasm. Rapid Heme Panel (RHP) is a custom, 95-gene, amplicon-based NGS panel (PMID: 27339098) that was launched at the Center for Advanced Molecular Diagnostics (CAMD), Brigham and Women's Hospital and Dana-Farber Cancer Institute in August of 2014. RHP covers a total territory of ~200 KB, including hotspots of oncogenes and whole coding exons of tumor suppressor genes that are frequently mutated in myeloid and lymphoid malignancies. Single nucleotide variants, insertions/deletions up to 52-bp, and copy number variations are detected. In the two years following its launch, over 5,000 RHP was performed and reported with an average turnaround time of 7.2 days from time of receipt into the lab. The specimen failed rate is <0.5% and sample repeat rate is <0.2%. Over half of the specimens came from patients with a known myeloid disease: 25% with acute leukemia, 15% with a myelodysplastic syndrome (MDS), 10% with a myeloproliferative neoplasm (MPN) and a minor fraction each with a variety of other myeloid neoplasms such as paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia, systemic mastocytosis or chronic myeloid leukemia (CML). Twenty percent of the specimens came from patients with known lymphoid malignancies such as hairy cell leukemia, chronic lymphocytic leukemia, lymphoplasmacytic lymphoma, or splenic marginal zone lymphoma. The remainder 25-30% of the specimens came from patients with abnormal blood count (CBC) such as anemia, neutropenia, thrombocytopenia, leukocytosis, thrombocytosis and/or abnormal serum protein electrophoresis (SPEP) where a myeloid or a lymphoid neoplasm was suspected. Greater than 98% of the time, the test was ordered by a hematologist/oncologist. Among patients with a prior diagnosis, >80% of them had at least one pathogenic alterations identified by RHP while about 30% of the patients with abnormal CBC or abnormal SPEP had positive findings. RHP results have been used to (1) provide eligibility for enrollment into clinical trials of targeted therapies; (2) monitor effect of therapy by quantifying variant allele fraction; (3) identify disease progression with detection of emergence of new variants; (4) evaluate post-transplant status by following allele fractions of pre-transplant pathogenic variants; (5) shorten the time and cost to diagnosis by establishing clonality and identification of disease-defining alterations. Disclosures No relevant conflicts of interest to declare.

Use of cytology centrifuged supernatants improves cost and turnaround time for targeted next generation sequencing

Diagnostic Cytopathology ◽

10.1002/dc.24548 ◽

2020 ◽

Vol 48 (12) ◽

pp. 1167-1172 ◽

Cited By ~ 1

Author(s):

HamzaN. Gokozan ◽

Aparna Harbhajanka ◽

Philip Bomeisl ◽

Claire W. Michael ◽

Navid Sadri

Keyword(s):

Turnaround Time ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Parkour LIMS: facilitating high-quality sample preparation in next generation sequencing

10.1101/338533 ◽

2018 ◽

Author(s):

E. Anatskiy ◽

D.P. Ryan ◽

B. Grüning ◽

L. Arrigoni ◽

T. Manke ◽

...

Keyword(s):

Sample Preparation ◽

Web Application ◽

Quality Criteria ◽

Turnaround Time ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Report Generation ◽

AbstractSummaryThis paper presents Parkour, a software package for sample processing and quality management of next generation sequencing data and samples. Starting with user requests, Parkour allows tracking and assessing samples based on predefined quality criteria through different stages of the sample preparation workflow. Ideally suited for academic core laboratories, the software aims to maximize efficiency and reduce turnaround time by intelligent sample grouping and a clear assignment of staff to work units. Tools for automated invoicing, interactive statistics on facility usage and simple report generation minimize administrative tasks. Provided as a web application, Parkour is a convenient tool for both deep sequencing service users and laboratory personal. A set of web APIs allow coordinated information sharing with local and remote bioinformaticians. The flexible structure allows workflow customization and simple addition of new features as well as the expansion to other domains.Availability and implementationThe code and documentation are available at https://github.com/maxplanck-ie/[email protected]

Development, Implementation and Assessment of Molecular Diagnostics by Next Generation Sequencing in Personalized Treatment of Cancer: Experience of a Public Reference Healthcare Hospital

Cancers ◽

10.3390/cancers11081196 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1196 ◽

Cited By ~ 3

Author(s):

Simarro ◽

Murria ◽

Pérez-Simó ◽

Llop ◽

Mancheño ◽

...

Keyword(s):

Single Gene ◽

Turnaround Time ◽

Genetic Alterations ◽

Molecular Diagnostic ◽

Public Healthcare ◽

Next Generation ◽

Primary Resistance ◽

Iso 15189 ◽

The establishment of precision medicine in cancer patients requires the study of several biomarkers. Single-gene testing approaches are limited by sample availability and turnaround time. Next generation sequencing (NGS) provides an alternative for detecting genetic alterations in several genes with low sample requirements. Here we show the implementation to routine diagnostics of a NGS assay under International Organization for Standardization (UNE-EN ISO 15189:2013) accreditation. For this purpose, 106 non-small cell lung cancer (NSCLC) and 102 metastatic colorectal cancer (mCRC) specimens were selected for NGS analysis with Oncomine Solid Tumor (ThermoFisher). In NSCLC the most prevalently mutated gene was TP53 (49%), followed by KRAS (31%) and EGFR (13%); in mCRC, TP53 (50%), KRAS (48%) and PIK3CA (16%) were the most frequently mutated genes. Moreover, NGS identified actionable genetic alterations in 58% of NSCLC patients, and 49% of mCRC patients did not harbor primary resistance mechanisms to anti-EGFR treatment. Validation with conventional approaches showed an overall agreement >90%. Turnaround time and cost analysis revealed that NGS implementation is feasible in the public healthcare context. Therefore, NGS is a multiplexed molecular diagnostic tool able to overcome the limitations of current molecular diagnosis in advanced cancer, allowing an improved and economically sustainable molecular profiling.