scholarly journals Locating a transgene integration site by nanopore sequencing

2019 ◽  
Author(s):  
Peter K. Nicholls ◽  
Daniel W. Bellott ◽  
Ting-Jan Cho ◽  
Tatyana Pyntikova ◽  
David C. Page
Keyword(s):  
Germ Line ◽  
Integration Site ◽  
Cost Effective ◽  
Chromosome 9 ◽  
Biological Research ◽  
Nanopore Sequencing ◽  
Fluorescent Reporter ◽  
Transgene Integration ◽  
Integration Sites ◽  
Foreign Dna

AbstractThe introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnm (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

scholarly journals Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform

2019 ◽  
Author(s):  
Ping Zhang ◽  
Devika Ganesamoorthy ◽  
Son Hoang Nguyen ◽  
Raymond Au ◽  
Lachlan J. Coin ◽  
...  
Keyword(s):  
Integration Site ◽  
Cost Effective ◽  
Restriction Enzyme Digestion ◽  
Nanopore Sequencing ◽  
Site Analysis ◽  
Minimum Capital ◽  
Vector Integration ◽  
Integration Site Analysis ◽  
Integration Sites

AbstractVector integration site analysis can be important in the follow-up of patients who received gene-modified cells, but current platforms based on next-generation sequencing are expensive and relatively inaccessible. We analyzed polyclonal T cells transduced by a gammaretroviral vector, SFG.iCasp9.2A.ΔCD19, from a clinical trial. Following restriction enzyme digestion, the unknown flanking genomic sequences were amplified by inverse polymerase chain reaction (PCR) or cassette ligation PCR. Nanopore sequencing could identify thousands of unique integration sites within polyclonal samples, with cassette ligation PCR showing less bias. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis.

427 INTEGRATION SITE ANALYSIS IN TRANSGENIC PIGS BY THERMAL ASYMMETRIC INTERLACED (TAIL)-PCR AND JUNCTION PCR

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
Q. R. Kong ◽  
Z. H. Liu
Keyword(s):  
Integration Site ◽  
Transgenic Animals ◽  
Pcr Analysis ◽  
Attractive Alternative ◽  
Transgenic Pigs ◽  
Degenerate Primers ◽  
Transgene Integration ◽  
Integration Sites ◽  
Specific Amplification ◽  
Tail Pcr

Transgenic animals have been used to study gene function, produce important proteins, xenotransplantation donor, and generate models for the study of human diseases. Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). However, when transgenic animals are produced by SCNT using randomly transfected cells as donor, the integration sites of transgene cannot be predicted. Many methods on the basis of genome walking have been demonstrated to clone transgene integration sites but they are either complicated or inefficient. In the study, we report a PCR-based method, thermal asymmetric interlaced PCR (TAIL-PCR), which relies on a series of 3 nested PCR reactions with transgene specific, designed with melting temperature of about 64, and arbitrary degenerate primers, by control of annealing temperature to efficiently reduce the nonspecific amplification to clone the integration sites in transgenic pigs by SCNT. Junction PCR combined with transgene-specific and integration site primers was performed to confirm the integration sites. Three integration sites were found (1 mapped on chromosome 4; the other 2 met a significant match in the pig expressed sequence tag database) in 2 founder transgenic pigs. Junction PCR resulted in specific amplification bands to identify the integration sites, and segregation of the integration sites was also detected in subsequent progeny by junction PCR analysis. We also used junction PCR combining with transgene-specific 5′ and 3′ integration site primers to analyze zygosity of the integration sites. Besides the specific amplification bands amplifying by transgene specific and integration site primers, bands amplified by 5′ and 3′ integration site primers were obtained to determine the heterozygosity of integration site. In conclusion, this strategy can be efficiently employed to clone transgene integration site and determine zygosity. This work was supported by grant from the State Transgenic Research Programme of China (Grant No. 2008ZX08006-002).

scholarly journals Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

2020 ◽  
Vol 8 (1) ◽  
pp. e000299
Author(s):  
Ping Zhang ◽  
Devika Ganesamoorthy ◽  
Son Hoang Nguyen ◽  
Raymond Au ◽  
Lachlan J Coin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Cost Effective ◽  
Inverse Pcr ◽  
Nanopore Sequencing ◽  
Next Generation ◽  
Short Read ◽  
Vector Integration ◽  
Integration Sites ◽  
Generation Sequencing

BackgroundAnalysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs.MethodsWe used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters,NcoIandBspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters.ResultsBoth inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3′long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing.ConclusionOur study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

scholarly journals Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing

2019 ◽  
Vol 9 (5) ◽  
pp. 1481-1486 ◽  
Author(s):  
Peter K. Nicholls ◽  
Daniel W. Bellott ◽  
Ting-Jan Cho ◽  
Tatyana Pyntikova ◽  
David C. Page
Keyword(s):  
Integration Site ◽  
Nanopore Sequencing ◽  
Transgene Integration

Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase

2000 ◽  
Vol 24 (6) ◽  
pp. 713-723 ◽  
Author(s):  
Rita Abranches ◽  
Ana P. Santos ◽  
Eva Wegel ◽  
Sarah Williams ◽  
Alexandra Castilho ◽  
...  
Keyword(s):  
Transgene Integration ◽  
Integration Sites

FISH Analysis of 142 EGFP Transgene Integration Sites into the Mouse Genome

Genomics ◽  
2002 ◽  
Vol 80 (6) ◽  
pp. 564-574 ◽  
Author(s):  
Tomoko Nakanishi ◽  
Asato Kuroiwa ◽  
Shuichi Yamada ◽  
Ayako Isotani ◽  
Atsuko Yamashita ◽  
...  
Keyword(s):  
Mouse Genome ◽  
Fish Analysis ◽  
Transgene Integration ◽  
Integration Sites

scholarly journals High resolution copy number inference in cancer using short-molecule nanopore sequencing

2020 ◽  
Author(s):  
Timour Baslan ◽  
Sam Kovaka ◽  
Fritz J. Sedlazeck ◽  
Yanming Zhang ◽  
Robert Wappel ◽  
...  
Keyword(s):  
Copy Number ◽  
Cost Effective ◽  
Chromosome Analysis ◽  
Ease Of Use ◽  
Precision Oncology ◽  
Nanopore Sequencing ◽  
Dna Molecules ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing

ABSTRACTGenome copy number is an important source of genetic variation in health and disease. In cancer, clinically actionable Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in terms of the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms. This represents a challenge for applications that require high read counts such as CNA inference. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that sequencing short DNA molecules reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for the sequencing of relatively short DNA molecules on nanopore devices with applications in research and medicine, that include but are not limited to, CNAs.

scholarly journals A New Rapid and Cost-Effective Method for Detection of Phages, ICEs and Virulence Factors Encoded by Streptococcus pyogenes

2011 ◽  
Vol 60 (3) ◽  
pp. 187-201 ◽  
Author(s):  
ANNA L. BOREK ◽  
JOANNA WILEMSKA ◽  
RADOSŁAW IZDEBSKI ◽  
WALERIA HRYNIEWICZ ◽  
IZABELA SITKIEWICZ
Keyword(s):  
Virulence Factors ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Effective System ◽  
Genes Encoding ◽  
Invasive Infections ◽  
Integration Sites ◽  
Severity Of The Disease

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.

scholarly journals HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
E Paietta ◽  
RJ Stockert ◽  
T Calvelli ◽  
P Papenhausen ◽  
SV Seremetis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Germ Line ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Chromosome 9 ◽  
Terminal Transferase

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58- kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony- stimulating factor (GM-CSF), as found in HL-60, is not observed in HL- T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface- antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4- , TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

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