CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing

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).

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RAISING: a high-performance method for identifying random transgene integration sites

10.21203/rs.3.rs-526444/v1 ◽

2021 ◽

Author(s):

Yusaku Wada ◽

Tomoo Sato ◽

Hiroo Hasegawa ◽

Takahiro Matsudaira ◽

Naganori Nao ◽

...

Keyword(s):

High Performance ◽

Viral Vectors ◽

Viral Infections ◽

Therapeutic Interventions ◽

Transgene Integration ◽

Adult T Cell Leukemia ◽

Integration Sites ◽

Infected Cells ◽

Novel Method ◽

Rapid Amplification

Abstract Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

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sppIDer: a species identification tool to investigate hybrid genomes with high-throughput sequencing

10.1101/333815 ◽

2018 ◽

Cited By ~ 1

Author(s):

Quinn K. Langdon ◽

David Peris ◽

Brian Kyle ◽

Chris Todd Hittinger

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rapid Identification ◽

Sequencing Data ◽

Pure Species ◽

High Throughput Sequencing Data ◽

Interspecies Hybrids ◽

Evolutionary Trajectories ◽

Low Coverage ◽

Reference Genomes

AbstractThe genomics era has expanded our knowledge about the diversity of the living world, yet harnessing high-throughput sequencing data to investigate alternative evolutionary trajectories, such as hybridization, is still challenging. Here we present sppIDer, a pipeline for the characterization of interspecies hybrids and pure species,that illuminates the complete composition of genomes. sppIDer maps short-read sequencing data to a combination genome built from reference genomes of several species of interest and assesses the genomic contribution and relative ploidy of each parental species, producing a series of colorful graphical outputs ready for publication. As a proof-of-concept, we use the genus Saccharomyces to detect and visualize both interspecies hybrids and pure strains, even with missing parental reference genomes. Through simulation, we show that sppIDer is robust to variable reference genome qualities and performs well with low-coverage data. We further demonstrate the power of this approach in plants, animals, and other fungi. sppIDer is robust to many different inputs and provides visually intuitive insight into genome composition that enables the rapid identification of species and their interspecies hybrids. sppIDer exists as a Docker image, which is a reusable, reproducible, transparent, and simple-to-run package that automates the pipeline and installation of the required dependencies (https://github.com/GLBRC/sppIDer).

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Locating a transgene integration site by nanopore sequencing

10.1101/520627 ◽

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.

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Fish analysis of multiple transgene integration sites in a beta casein-antithrombin III goat line

Theriogenology ◽

10.1016/s0093-691x(98)90751-8 ◽

1998 ◽

Vol 49 (1) ◽

pp. 398 ◽

Cited By ~ 3

Author(s):

J. Williams ◽

F.A.Ponce de Leon ◽

P. Midura ◽

M. Hamngton ◽

H. Meade ◽

...

Keyword(s):

Antithrombin Iii ◽

Fish Analysis ◽

Transgene Integration ◽

Integration Sites ◽

Beta Casein

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Assembly of the Mitochondrial Genome in the Campanulaceae Family Using Illumina Low-Coverage Sequencing

Genes ◽

10.3390/genes9080383 ◽

2018 ◽

Vol 9 (8) ◽

pp. 383 ◽

Cited By ~ 2

Author(s):

Hyun-Oh Lee ◽

Ji-Weon Choi ◽

Jeong-Ho Baek ◽

Jae-Hyeon Oh ◽

Sang-Choon Lee ◽

...

Keyword(s):

Mitochondrial Genome ◽

Dna Sequencing ◽

De Novo ◽

Genome Structure ◽

Sequencing Data ◽

Phylogenetic Characterization ◽

Low Coverage ◽

Paired End Sequencing ◽

Circular Chromosomes

Platycodon grandiflorus (balloon flower) and Codonopsis lanceolata (bonnet bellflower) are important herbs used in Asian traditional medicine, and both belong to the botanical family Campanulaceae. In this study, we designed and implemented a de novo DNA sequencing and assembly strategy to map the complete mitochondrial genomes of the first two members of the Campanulaceae using low-coverage Illumina DNA sequencing data. We produced a total of 28.9 Gb of paired-end sequencing data from the genomic DNA of P. grandiflorus (20.9 Gb) and C. lanceolata (8.0 Gb). The assembled mitochondrial genome of P. grandiflorus was found to consist of two circular chromosomes; the master circle contains 56 genes, and the minor circle contains 42 genes. The C. lanceolata mitochondrial genome consists of a single circle harboring 54 genes. Using a comparative genome structure and a pattern of repeated sequences, we show that the P. grandiflorus minor circle resulted from a recombination event involving the direct repeats of the master circle. Our dataset will be useful for comparative genomics and for evolutionary studies, and will facilitate further biological and phylogenetic characterization of species in the Campanulaceae.

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