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Article ; Online: Rapid microdissection of tissue sections via laser ablation.

Coope, Robin Jn / Pleasance, Stephen / Pandoh, Pawan / Schlosser, Colin / Corbett, Richard D / Marra, Marco A

2024

Abstract: We demonstrate a method for tissue microdissection using scanning laser ablation that is approximately two orders of magnitude faster than conventional laser capture microdissection. Our novel approach uses scanning laser optics and a slide coating under ...

Abstract	We demonstrate a method for tissue microdissection using scanning laser ablation that is approximately two orders of magnitude faster than conventional laser capture microdissection. Our novel approach uses scanning laser optics and a slide coating under the tissue that can be excited by the laser to selectively eject regions of tissue for further processing. Tissue was dissected at 0.117 s/mm
Language	English
Publishing date	2024-03-01
Publishing country	England
Document type	Journal Article
ZDB-ID	80261-x
ISSN	1472-4146 ; 0021-9746
ISSN (online)	1472-4146
ISSN	0021-9746
DOI	10.1136/jcp-2023-209361
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Article ; Online: Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

Gadkar, Vijay J / Goldfarb, David M / Al-Rawahi, Ghada N / Srigley, Jocelyn A / Smailus, Duane E / Coope, Robin J N / Pleasance, Stephen / Watson, Nicole / Chen, Tammy / Lam, Sunny / Hoang, Linda / Tilley, Peter A G

Scientific reports

2023 Volume 13, Issue 1, Page(s) 4241

Abstract: As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput ... ...

Abstract	As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.
MeSH term(s)	Humans ; SARS-CoV-2/genetics ; COVID-19/diagnosis ; Pandemics ; COVID-19 Testing ; Specimen Handling ; Multiplex Polymerase Chain Reaction ; Sensitivity and Specificity ; RNA, Viral/analysis
Chemical Substances	RNA, Viral
Language	English
Publishing date	2023-03-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-30993-2
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Article ; Online: Complete Mitochondrial Genome of a Gymnosperm, Sitka Spruce (Picea sitchensis), Indicates a Complex Physical Structure.

Jackman, Shaun D / Coombe, Lauren / Warren, René L / Kirk, Heather / Trinh, Eva / MacLeod, Tina / Pleasance, Stephen / Pandoh, Pawan / Zhao, Yongjun / Coope, Robin J / Bousquet, Jean / Bohlmann, Joerg / Jones, Steven J M / Birol, Inanc

Genome biology and evolution

2020 Volume 12, Issue 7, Page(s) 1174–1179

Abstract: Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, ... ...

Abstract	Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the complete 5.5-Mb mitochondrial genome of Sitka spruce (Picea sitchensis), to date, one of the largest mitochondrial genomes of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads based on sequence homology to the white spruce mitochondrial genome. The assembly graph shows a multipartite genome structure, composed of one smaller 168-kb circular segment of DNA, and a larger 5.4-Mb single component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.
Language	English
Publishing date	2020-05-25
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1759-6653
ISSN (online)	1759-6653
DOI	10.1093/gbe/evaa108
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Article ; Online: Whole-slide laser microdissection for tumour enrichment.

Coope, Robin Jn / Schlosser, Colin / Corbett, Richard D / Pleasance, Stephen / Tessier-Cloutier, Basile / Pandoh, Pawan / Kirk, Heather / Haile, Simon / Zhao, Yongjun / Mungall, Andrew J / Marra, Marco A

The Journal of pathology

2020 Volume 253, Issue 2, Page(s) 225–233

Abstract: The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded ... ...

Abstract	The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
MeSH term(s)	Animals ; Formaldehyde ; Humans ; Laser Capture Microdissection ; Liver/pathology ; Mice ; Mice, Inbred C57BL ; Neoplasms/pathology ; Precision Medicine ; Tissue Fixation
Chemical Substances	Formaldehyde (1HG84L3525)
Language	English
Publishing date	2020-12-09
Publishing country	England
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3119-7
ISSN	1096-9896 ; 0022-3417
ISSN (online)	1096-9896
ISSN	0022-3417
DOI	10.1002/path.5575
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Article: Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

Coope, Robin J N / Matic, Nancy / Pandoh, Pawan K / Corbett, Richard D / Smailus, Duane E / Pleasance, Stephen / Lowe, Christopher F / Ritchie, Gordon / Chorlton, Samuel D / Young, Matthew / Ally, Adrian A / Asano, Jennifer K / Carlsen, Rebecca E / Chahal, Sundeep S / Zhao, Yongjun / Holmes, Daniel T / Romney, Marc G / Jones, Steven J M / Marra, Marco A

The journal of applied laboratory medicine

2022 Volume 7, Issue 5, Page(s) 1025–1036

Abstract: Background: To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow.: Methods: We used the established ... ...

Abstract	Background: To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow. Methods: We used the established ARTIC protocol with approximately 400 bp amplicons sequenced on Oxford Nanopore's MinION. Sequences were analyzed using Nextclade, assigning both a clade and quality score to each sample. Results: A total of 2179 samples on twenty-five 96-well plates were sequenced. Plates of purified RNA were processed within 12 h, sequencing required up to 24 h, and analysis of each pooled plate required 1 h. The use of samples with known threshold cycle (Ct) values enabled normalization, acted as a quality control check, and revealed a strong correlation between sample Ct values and successful analysis, with 85% of samples with Ct < 30 achieving a "good" Nextclade score. Less abundant samples responded to enrichment with the fraction of Ct > 30 samples achieving a "good" classification rising by 60% after addition of a post-ARTIC PCR normalization. Serial dilutions of 3 variant of concern samples, diluted from approximately Ct = 16 to approximately Ct = 50, demonstrated successful sequencing to Ct = 37. The sample set contained a median of 24 mutations per sample and a total of 1281 unique mutations with reduced sequence read coverage noted in some regions of some samples. A total of 10 separate strains were observed in the sample set, including 3 variants of concern prevalent in British Columbia in the spring of 2021. Conclusions: We demonstrated a robust automated sequencing pipeline that takes advantage of input Ct values to improve reliability.
MeSH term(s)	COVID-19/diagnosis ; COVID-19/epidemiology ; Humans ; Nanopore Sequencing ; Nanopores ; Reproducibility of Results ; SARS-CoV-2/genetics
Language	English
Publishing date	2022-06-20
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2576-9456
ISSN	2576-9456
DOI	10.1093/jalm/jfac054
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Article ; Online: MAVIS: merging, annotation, validation, and illustration of structural variants

Reisle, Caralyn / Mungall, Karen L. / Choo, Caleb / Paulino, Daniel / Bleile, Dustin W. / Muhammadzadeh, Amir / Mungall, Andrew J. / Moore, Richard A. / Shlafman, Inna / Coope, Robin / Pleasance, Stephen / Ma, Yussanne / Jones, Steven J. M.

Bioinformatics. 2019 Feb. 01, v. 35, no. 3, p. 515-517

2019 , Page(s) 515–517

Abstract: Reliably identifying genomic rearrangements and interpreting their impact is a key step in understanding their role in human cancers and inherited genetic diseases. Many short read algorithmic approaches exist but all have appreciable false negative ... ...

Abstract	Reliably identifying genomic rearrangements and interpreting their impact is a key step in understanding their role in human cancers and inherited genetic diseases. Many short read algorithmic approaches exist but all have appreciable false negative rates. A common approach is to evaluate the union of multiple tools increasing sensitivity, followed by filtering to retain specificity. Here we describe an application framework for the rapid generation of structural variant consensus, unique in its ability to visualize the genetic impact and context as well as process both genome and transcriptome data. http://mavis.bcgsc.ca Supplementary data are available at Bioinformatics online.
Keywords	bioinformatics ; genome ; genomics ; humans ; transcriptome
Language	English
Dates of publication	2019-0201
Size	p. 515-517
Publishing place	Oxford University Press
Document type	Article ; Online
ZDB-ID	1468345-3
ISSN	1367-4811 ; 1460-2059
ISSN	1367-4811 ; 1460-2059
DOI	10.1093/bioinformatics/bty621
Database	NAL-Catalogue (AGRICOLA)

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Article: Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents

Haile, Simon / Nikiforuk, Aidan M. / Pandoh, Pawan K. / Twa, David D.W. / Smailus, Duane E. / Nguyen, Jason / Pleasance, Stephen / Wong, Angus / Zhao, Yongjun / Eisler, Diane / Moksa, Michelle / Cao, Qi / Wong, Marcus / Su, Edmund / Krzywinski, Martin / Nelson, Jessica / Mungall, Andrew J. / Tsang, Frankie / Prentice, Leah M. /

Jassem, Agatha / Manges, Amee R. / Jones, Steven J.M. / Coope, Robin J. / Prystajecky, Natalie / Marra, Marco A. / Krajden, Mel / Hirst, Martin

Journal of virological methods. 2022 Jan., v. 299

2022

Abstract: The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. ... ...

Abstract	The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.
Keywords	COVID-19 infection ; Severe acute respiratory syndrome coronavirus 2 ; liquids ; magnetism ; nucleic acids
Language	English
Dates of publication	2022-01
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2021.114339
Database	NAL-Catalogue (AGRICOLA)

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Article: A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

Frontiers in genetics

2021 Volume 12, Page(s) 665888

Abstract: RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the ... ...

Abstract	RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.
Language	English
Publishing date	2021-06-03
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2606823-0
ISSN	1664-8021
ISSN	1664-8021
DOI	10.3389/fgene.2021.665888
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Article ; Online: MAVIS: merging, annotation, validation, and illustration of structural variants.

Reisle, Caralyn / Mungall, Karen L / Choo, Caleb / Paulino, Daniel / Bleile, Dustin W / Muhammadzadeh, Amir / Mungall, Andrew J / Moore, Richard A / Shlafman, Inna / Coope, Robin / Pleasance, Stephen / Ma, Yussanne / Jones, Steven J M

Bioinformatics (Oxford, England)

2017 Volume 35, Issue 3, Page(s) 515–517

Abstract: Summary: Reliably identifying genomic rearrangements and interpreting their impact is a key step in understanding their role in human cancers and inherited genetic diseases. Many short read algorithmic approaches exist but all have appreciable false ... ...

Abstract	Summary: Reliably identifying genomic rearrangements and interpreting their impact is a key step in understanding their role in human cancers and inherited genetic diseases. Many short read algorithmic approaches exist but all have appreciable false negative rates. A common approach is to evaluate the union of multiple tools increasing sensitivity, followed by filtering to retain specificity. Here we describe an application framework for the rapid generation of structural variant consensus, unique in its ability to visualize the genetic impact and context as well as process both genome and transcriptome data. Availability and implementation: http://mavis.bcgsc.ca. Supplementary information: Supplementary data are available at Bioinformatics online.
MeSH term(s)	Computational Biology ; Genomics ; Humans ; Neoplasms/genetics ; Software ; Transcriptome
Language	English
Publishing date	2017-11-02
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/bty621
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Zs.A 2374: Show issues

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Article: Co-occurrence of anaerobic bacteria in colorectal carcinomas.

Warren, René L / Freeman, Douglas J / Pleasance, Stephen / Watson, Peter / Moore, Richard A / Cochrane, Kyla / Allen-Vercoe, Emma / Holt, Robert A

Microbiome

2013 Volume 1, Issue 1, Page(s) 16

Abstract: Background: Numerous cancers have been linked to microorganisms. Given that colorectal cancer is a leading cause of cancer deaths and the colon is continuously exposed to a high diversity of microbes, the relationship between gut mucosal microbiome and ... ...

Abstract	Background: Numerous cancers have been linked to microorganisms. Given that colorectal cancer is a leading cause of cancer deaths and the colon is continuously exposed to a high diversity of microbes, the relationship between gut mucosal microbiome and colorectal cancer needs to be explored. Metagenomic studies have shown an association between Fusobacterium species and colorectal carcinoma. Here, we have extended these studies with deeper sequencing of a much larger number (n = 130) of colorectal carcinoma and matched normal control tissues. We analyzed these data using co-occurrence networks in order to identify microbe-microbe and host-microbe associations specific to tumors. Results: We confirmed tumor over-representation of Fusobacterium species and observed significant co-occurrence within individual tumors of Fusobacterium, Leptotrichia and Campylobacter species. This polymicrobial signature was associated with over-expression of numerous host genes, including the gene encoding the pro-inflammatory chemokine Interleukin-8. The tumor-associated bacteria we have identified are all Gram-negative anaerobes, recognized previously as constituents of the oral microbiome, which are capable of causing infection. We isolated a novel strain of Campylobacter showae from a colorectal tumor specimen. This strain is substantially diverged from a previously sequenced oral Campylobacter showae isolate, carries potential virulence genes, and aggregates with a previously isolated tumor strain of Fusobacterium nucleatum. Conclusions: A polymicrobial signature of Gram-negative anaerobic bacteria is associated with colorectal carcinoma tissue.
Language	English
Publishing date	2013-05-15
Publishing country	England
Document type	Journal Article
ZDB-ID	2697425-3
ISSN	2049-2618
ISSN	2049-2618
DOI	10.1186/2049-2618-1-16
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