scholarly journals Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

2018 ◽  
Author(s):  
Mudra Hegde ◽  
Christine Strand ◽  
Ruth E. Hanna ◽  
John G. Doench
Keyword(s):  
Next Generation Sequencing ◽  
Genomic Dna ◽  
Mammalian Cells ◽  
Pcr Amplification ◽  
Next Generation ◽  
Dna Template ◽  
Template Dna ◽  
Generation Sequencing

ABSTRACTMany implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

scholarly journals Development of microsatellite markers from genomic DNA of Parashorea malaanonan (Dipterocarpaceae) using next-generation sequencing

2019 ◽  
Vol 68 (1) ◽  
pp. 22-25 ◽  
Author(s):  
Crusty E. Tinio ◽  
Saneyoshi Ueno ◽  
Kentaro Uchiyama ◽  
Lerma S. J. Maldia ◽  
Nobuhiro Tomaru
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Markers ◽  
Genomic Dna ◽  
Molecular Ecology ◽  
Pcr Amplification ◽  
The Philippines ◽  
Next Generation ◽  
Adult Trees ◽  
High Level ◽  
Generation Sequencing

Abstract Twenty polymorphic microsatellite markers were developed, using Next Generation Sequencing (Illumina), from genomic DNA of Parashorea malaanonan, a species of the Dipterocarpa­ceae which is ecologically and economically important in the Philippines. Thirty adult trees from a natural population were used to assess the success of PCR amplification and the degree of polymorphism. The number of alleles per locus varied from three to 13, and observed and expected heterozygosity varied from 0.200 to 0.808 and from 0.301 to 0.890 respectively. Total exclusion probabilities for the first and second parents over the 20 loci were 0.99932499 and 0.99999723 respectively. The high level of polymorphism at these loci makes it possible to obtain precise estimates of genetic parameters and thus the markers will help in studies on population genetics, conservation gene­tics, and molecular ecology of P. malaanonan.

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 Detection of Listeria monocytogenes in a patient with meningoencephalitis using next-generation sequencing: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zi-Wei Lan ◽  
Min-Jia Xiao ◽  
Yuan-lin Guan ◽  
Ya-Jing Zhan ◽  
Xiang-Qi Tang
Keyword(s):  
Listeria Monocytogenes ◽  
Next Generation Sequencing ◽  
Mammalian Cells ◽  
Clinical Manifestations ◽  
Bacterial Pathogen ◽  
Next Generation ◽  
Accurate Treatment ◽  
The Central Nervous System ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background Listeria monocytogenes (L. monocytogenes) is a facultative intracellular bacterial pathogen which can invade different mammalian cells and reach to the central nervous system (CNS), leading to meningoencephalitis and brain abscesses. In the diagnosis of L. monocytogenes meningoencephalitis (LMM), the traditional test often reports negative owing to the antibiotic treatment or a low number of bacteria in the cerebrospinal fluid. To date, timely diagnosis and accurate treatment remains a challenge for patients with listeria infections. Case presentation We present the case of a 66-year-old woman whose clinical manifestations were suspected as tuberculous meningoencephalitis, but the case was finally properly diagnosed as LMM by next-generation sequencing (NGS). The patient was successfully treated using a combined antibacterial therapy, comprising ampicillin and trimethoprim-sulfamethoxazole. Conclusion To improve the sensitivity of LMM diagnosis, we used NGS for the detection of L. monocytogenes. Hence, the clinical utility of this approach can be very helpful since it provides quickly and trust results.

Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples

2015 ◽  
Vol 407 (7) ◽  
pp. 1841-1848 ◽  
Author(s):  
Marta Pawluczyk ◽  
Julia Weiss ◽  
Matthew G. Links ◽  
Mikel Egaña Aranguren ◽  
Mark D. Wilkinson ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Quantitative Evaluation ◽  
Pcr Amplification ◽  
Next Generation ◽  
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

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.

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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