scholarly journals Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

10.3791/53840 ◽  
2016 ◽  
Author(s):  
Erik Serrao ◽  
Peter Cherepanov ◽  
Alan N. Engelman
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
Retroviral Integration ◽  
Dna Mapping ◽  
Integration Sites ◽  
Generation Sequencing

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 Targeted enrichment of genomic DNA regions for next-generation sequencing

2011 ◽  
Vol 10 (6) ◽  
pp. 374-386 ◽  
Author(s):  
F. Mertes ◽  
A. ElSharawy ◽  
S. Sauer ◽  
J. M. L. M. van Helvoort ◽  
P. J. van der Zaag ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Next Generation ◽  
Generation Sequencing ◽  
Targeted Enrichment

scholarly journals VirusSeq: software to identify viruses and their integration sites using next-generation sequencing of human cancer tissue

2012 ◽  
Vol 29 (2) ◽  
pp. 266-267 ◽  
Author(s):  
Yunxin Chen ◽  
Hui Yao ◽  
Erika J. Thompson ◽  
Nizar M. Tannir ◽  
John N. Weinstein ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Human Cancer ◽  
Cancer Tissue ◽  
Next Generation ◽  
Integration Sites ◽  
Human Cancer Tissue ◽  
Generation Sequencing

scholarly journals Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

Genes ◽  
2014 ◽  
Vol 5 (4) ◽  
pp. 1115-1131 ◽  
Author(s):  
Yukio Kurihara ◽  
Yuko Makita ◽  
Mika Kawashima ◽  
Hidefumi Hamasaki ◽  
Yoshiharu Yamamoto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Dna Fragments ◽  
Next Generation ◽  
Regulatory Potential ◽  
Generation Sequencing

Genomic alteration features of six cancers and their correlation with clinical characters using the next generation sequencing.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13130-e13130
Author(s):  
Qian Dong ◽  
Haiying Yang ◽  
Da Jiang
Keyword(s):  
Next Generation Sequencing ◽  
Family History ◽  
Mutation Rate ◽  
Genomic Dna ◽  
Genomic Alteration ◽  
Next Generation ◽  
Cancer History ◽  
Invasive Approach ◽  
Cancer Types ◽  
Generation Sequencing

e13130 Background: Next-generation sequencing (NGS) examination based on liquid biopsy has been agreed as a kind of routine diagnostic tool in cancer field and may provide new sights about oncogenesis mechanisms. This study aimed to observe the genomic alteration features of common cancers and their correlation with clinical characters. Methods: We enrolled 39 subjects (38 subjects with cancer and 1 healthy subject) and administrated mutation examination using a panel including 38 genes, which targeted 6 cancer types. For each subject, the genomic DNA was extracted and NGS was performed. Results: All patients were found to carry at least one alteration among the 38 genes. PSM2 showed a highest mutation rate, with 77% subjects carrying mutations in this gene. Besides, MSH2 and STK11, etc. exhibited a high mutation rate. Overall 17 genes were found to contain known pathogenic or outcome-unknown mutations, among which 15 had outcome-unknown mutations and 5 had recognized pathogenic mutations. RAD50 had a highest frequency of outcome-unknown mutations, and secondly MLH1 and MRE11A. The clinical characters including sex, metastasis/recurrence, and family cancer history had relationships with mutation types. Conclusions: Genomic DNA sequencing is a feasible and minimally invasive approach in cancer genetic analysis and a promising tool in prediction of cancer onset and prognosis. A large amount of variations are associated with sex, family history and cancer types, and may decide metastasis/recurrence outcomes. Those who have a family history of cancer are recommended to receive NGS examination.

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