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Article ; Online: High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Alegria, Andrew D / Joshi, Amey S / Mendana, Jorge Blanco / Khosla, Kanav / Smith, Kieran T / Auch, Benjamin / Donovan, Margaret / Bischof, John / Gohl, Daryl M / Kodandaramaiah, Suhasa B

Genetics

2024 Volume 226, Issue 4

Abstract: Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor- ... ...

Abstract	Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.
MeSH term(s)	Animals ; Humans ; Robotics ; Zebrafish/genetics ; Microinjections/methods ; Drosophila melanogaster/genetics ; Animals, Genetically Modified
Language	English
Publishing date	2024-02-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	2167-2
ISSN	1943-2631 ; 0016-6731
ISSN (online)	1943-2631
ISSN	0016-6731
DOI	10.1093/genetics/iyae025
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Article ; Online: A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2.

Gohl, Daryl M / Garbe, John / Grady, Patrick / Daniel, Jerry / Watson, Ray H B / Auch, Benjamin / Nelson, Andrew / Yohe, Sophia / Beckman, Kenneth B

BMC genomics

2020 Volume 21, Issue 1, Page(s) 863

Abstract: Background: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of ... ...

Abstract	Background: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Results: Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. Conclusions: The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.
MeSH term(s)	Benchmarking ; COVID-19/diagnosis ; COVID-19/epidemiology ; COVID-19/virology ; COVID-19 Nucleic Acid Testing/methods ; COVID-19 Nucleic Acid Testing/standards ; Genome, Viral/genetics ; Humans ; Molecular Epidemiology ; Mutation ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; SARS-CoV-2/isolation & purification ; Sequence Analysis/methods ; Sequence Analysis/standards
Chemical Substances	RNA, Viral
Language	English
Publishing date	2020-12-04
Publishing country	England
Document type	Journal Article
ISSN	1471-2164
ISSN (online)	1471-2164
DOI	10.1186/s12864-020-07283-6
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Article ; Online: Multiscale, multi-perspective imaging assisted robotic microinjection of 3D biological structures.

Joshi, Amey S / Alegria, Andrew D / Auch, Benjamin / Khosla, Kanav / Mendana, Jorge Blanco / Liu, Kunpeng / Bischof, John / Gohl, Daryl M / Kodandaramaiah, Suhasa B

Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference

2021 Volume 2021, Page(s) 4844–4850

Abstract: Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, ... ...

Abstract	Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.
MeSH term(s)	Animals ; Drosophila melanogaster ; Microinjections ; Robotic Surgical Procedures ; Robotics ; Zebrafish
Language	English
Publishing date	2021-12-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2694-0604
ISSN (online)	2694-0604
DOI	10.1109/EMBC46164.2021.9630858
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Article ; Online: Dissecting and tuning primer editing by proofreading polymerases.

Gohl, Daryl M / Auch, Benjamin / Certano, Amanda / LeFrançois, Brice / Bouevitch, Anne / Doukhanine, Evgueni / Fragel, Christina / Macklaim, Jean / Hollister, Emily / Garbe, John / Beckman, Kenneth B

Nucleic acids research

2021 Volume 49, Issue 15, Page(s) e87

Abstract: Proofreading polymerases have 3' to 5' exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the ...

Abstract	Proofreading polymerases have 3' to 5' exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.
MeSH term(s)	DNA Primers/genetics ; DNA Replication/genetics ; DNA-Directed DNA Polymerase/genetics ; Exonucleases/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Microbiota/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Ribosomal, 16S/genetics ; Skin/microbiology
Chemical Substances	DNA Primers ; RNA, Ribosomal, 16S ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; Exonucleases (EC 3.1.-)
Language	English
Publishing date	2021-06-14
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkab471
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Article ; Online: Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Forry, Samuel P / Servetas, Stephanie L / Kralj, Jason G / Soh, Keng / Hadjithomas, Michalis / Cano, Raul / Carlin, Martha / Amorim, Maria G de / Auch, Benjamin / Bakker, Matthew G / Bartelli, Thais F / Bustamante, Juan P / Cassol, Ignacio / Chalita, Mauricio / Dias-Neto, Emmanuel / Duca, Aaron Del / Gohl, Daryl M / Kazantseva, Jekaterina / Haruna, Muyideen T /

Menzel, Peter / Moda, Bruno S / Neuberger-Castillo, Lorieza / Nunes, Diana N / Patel, Isha R / Peralta, Rodrigo D / Saliou, Adrien / Schwarzer, Rolf / Sevilla, Samantha / Takenaka, Isabella K T M / Wang, Jeremy R / Knight, Rob / Gevers, Dirk / Jackson, Scott A

Scientific reports

2024 Volume 14, Issue 1, Page(s) 9785

Abstract: Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between ... ...

Abstract	Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.
MeSH term(s)	Humans ; Metagenomics/methods ; Metagenomics/standards ; RNA, Ribosomal, 16S/genetics ; Feces/microbiology ; Microbiota/genetics ; Bias ; Metagenome ; Gastrointestinal Microbiome/genetics ; Sequence Analysis, DNA/methods ; Bacteria/genetics ; Bacteria/classification ; Bacteria/isolation & purification ; High-Throughput Nucleotide Sequencing/methods
Chemical Substances	RNA, Ribosomal, 16S
Language	English
Publishing date	2024-04-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural ; Comparative Study
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-024-57981-4
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Article ; Online: AR gene rearrangement analysis in liquid biopsies reveals heterogeneity in lethal prostate cancer.

Daniel, Mark / Knutson, Todd P / Sperger, Jamie M / Li, Yingming / Singh, Anupama / Stahlfeld, Charlotte N / Passow, Courtney / Auch, Benjamin / Lang, Joshua M / Dehm, Scott M

Endocrine-related cancer

2021 Volume 28, Issue 9, Page(s) 645–655

Abstract: Castration-resistant prostate cancer (CRPC) is driven by AR gene aberrations that arise during androgen receptor (AR)-targeted therapy. AR amplification and mutations have been profiled in circulating tumor cells (CTCs), but whether AR gene ... ...

Abstract	Castration-resistant prostate cancer (CRPC) is driven by AR gene aberrations that arise during androgen receptor (AR)-targeted therapy. AR amplification and mutations have been profiled in circulating tumor cells (CTCs), but whether AR gene rearrangements can be assessed in CTCs is unknown. In this study, we leveraged CRPC cell lines with defined AR gene rearrangements to develop and validate a CTC DNA analysis approach that utilized whole genome amplification and targeted DNA-sequencing of AR and other genes important in CRPC. We tested the utility of this approach by analyzing matched CTC DNA and plasma cell-free DNA (cfDNA) from a case series of ten CRPC patients. One of ten CTC samples and two of ten cfDNA samples were positive for AR gene rearrangements. All AR gene rearrangements were discordant between matched liquid biopsy samples. One patient harbored separate AR gene rearrangements in CTC DNA and cfDNA, but concordant AR amplification and AR T878A mutation. This patient also displayed concordant loss of TP53 and PTEN, but the loss of RB1 in cfDNA only. The overall frequency of discordant alterations in these genes between matched CTC DNA and cfDNA was high. This study establishes the technical feasibility of analyzing structural rearrangements, mutations, and copy number variants in AR and other CRPC genes using two different sources of DNA from a single blood sample. Paired CTC DNA and cfDNA analysis may have utility for capturing the heterogeneity of genetic alterations in CRPC patients.
MeSH term(s)	Cell-Free Nucleic Acids ; Gene Rearrangement ; Humans ; Liquid Biopsy ; Male ; Neoplastic Cells, Circulating/metabolism ; Prostatic Neoplasms, Castration-Resistant/metabolism ; Receptors, Androgen/genetics
Chemical Substances	Cell-Free Nucleic Acids ; Receptors, Androgen
Language	English
Publishing date	2021-08-11
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1218450-0
ISSN	1479-6821 ; 1351-0088
ISSN (online)	1479-6821
ISSN	1351-0088
DOI	10.1530/ERC-21-0157
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Article ; Online: A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

Gohl, Daryl M. / Garbe, John / Grady, Patrick / Daniel, Jerry / Watson, Ray H. B. / Auch, Benjamin / Nelson, Andrew / Yohe, Sophia / Beckman, Kenneth B.

bioRxiv

Abstract: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are ... ...

Abstract	The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.
Keywords	covid19
Publisher	BioRxiv; WHO
Document type	Article ; Online
DOI	10.1101/2020.05.11.088724
Database	COVID19

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Article ; Online: Comparative analysis of genome-wide DNA methylation identifies patterns that associate with conserved transcriptional programs in osteosarcoma.

Mills, Lauren J / Scott, Milcah C / Shah, Pankti / Cunanan, Anne R / Deshpande, Archana / Auch, Benjamin / Curtin, Bridget / Beckman, Kenneth B / Spector, Logan G / Sarver, Aaron L / Subramanian, Subbaya / Richmond, Todd A / Modiano, Jaime F

Bone

2020 Volume 158, Page(s) 115716

Abstract: Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor protein p53 (TP53) is nearly universal other high frequency ...

Abstract	Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor protein p53 (TP53) is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. SIGNIFICANCE: Genome wide comparison of DNA methylation patterns in osteosarcoma across two species lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of varied mutational landscapes.
MeSH term(s)	Animals ; Bone Neoplasms/genetics ; DNA Methylation/genetics ; Dogs ; Epigenomics ; Genomics ; Mice ; Osteosarcoma/genetics ; Osteosarcoma/pathology
Language	English
Publishing date	2020-10-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	632515-4
ISSN	1873-2763 ; 8756-3282
ISSN (online)	1873-2763
ISSN	8756-3282
DOI	10.1016/j.bone.2020.115716
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Zs.A 1571: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article: Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification

Gohl, Daryl M / Magli, Alessandro / Garbe, John / Becker, Aaron / Johnson, Darrell M / Anderson, Shea / Auch, Benjamin / Billstein, Bradley / Froehling, Elyse / McDevitt, Shana L / Beckman, Kenneth B

Genome biology. 2019 Dec., v. 20, no. 1

2019

Abstract: Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, ... ...

Abstract	Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.
Keywords	droplets ; economics ; genomics ; high-throughput nucleotide sequencing ; plasmids ; polymerase chain reaction ; transgenes ; transposons
Language	English
Dates of publication	2019-12
Size	p. 85.
Publishing place	BioMed Central
Document type	Article
ZDB-ID	2040529-7
ISSN	1474-760X ; 1465-6906
ISSN (online)	1474-760X
ISSN	1465-6906
DOI	10.1186/s13059-019-1691-6
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Article ; Online: Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

Gohl, Daryl M / Magli, Alessandro / Garbe, John / Becker, Aaron / Johnson, Darrell M / Anderson, Shea / Auch, Benjamin / Billstein, Bradley / Froehling, Elyse / McDevitt, Shana L / Beckman, Kenneth B

Genome biology

2019 Volume 20, Issue 1, Page(s) 85

Abstract	Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.
MeSH term(s)	Animals ; DNA Restriction Enzymes ; Genetic Techniques ; Humans ; Sequence Tagged Sites
Chemical Substances	DNA Restriction Enzymes (EC 3.1.21.-)
Language	English
Publishing date	2019-04-29
Publishing country	England
Document type	Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2040529-7
ISSN	1474-760X ; 1474-760X
ISSN (online)	1474-760X
ISSN	1474-760X
DOI	10.1186/s13059-019-1691-6
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