Correction to: ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing

Abstract Background Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism’s genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. Results Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. Conclusions In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.

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Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Genes ◽

10.3390/genes11010092 ◽

2020 ◽

Vol 11 (1) ◽

pp. 92 ◽

Cited By ~ 1

Author(s):

Shannon J. McKie ◽

Anthony Maxwell ◽

Keir C. Neuman

Keyword(s):

Next Generation Sequencing ◽

Dna Cleavage ◽

Next Generation ◽

Dna Breaks ◽

Genome Wide ◽

Extension Activity ◽

Nucleotide Resolution ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

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Vision from next generation sequencing: Multi-dimensional genome-wide analysis for producing gene regulatory networks underlying retinal development, aging and disease

Progress in Retinal and Eye Research ◽

10.1016/j.preteyeres.2015.01.005 ◽

2015 ◽

Vol 46 ◽

pp. 1-30 ◽

Cited By ~ 35

Author(s):

Hyun-Jin Yang ◽

Rinki Ratnapriya ◽

Tiziana Cogliati ◽

Jung-Woong Kim ◽

Anand Swaroop

Keyword(s):

Next Generation Sequencing ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Retinal Development ◽

Next Generation ◽

Genome Wide Analysis ◽

Genome Wide ◽

Gene Regulatory ◽

Generation Sequencing

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A genomic approach to selecting robust and versatile SNP sets from next-generation sequencing data for genome-wide association study in citrus cultivars

Acta Horticulturae ◽

10.17660/actahortic.2016.1135.4 ◽

2016 ◽

pp. 23-32 ◽

Cited By ~ 7

Author(s):

T. Shimizu ◽

E. Kaminuma ◽

K. Nonaka ◽

T. Yoshioka ◽

S. Goto ◽

...

Keyword(s):

Next Generation Sequencing ◽

Association Study ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Genome Wide ◽

Genomic Approach ◽

Generation Sequencing

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Development of genome-wide simple sequence repeat markers in Codonopsis lanceolata using next-generation sequencing

Horticulture Environment and Biotechnology ◽

10.1007/s13580-021-00389-0 ◽

2021 ◽

Author(s):

Serim Kim ◽

Namsu Jo ◽

Jinsu Gil ◽

Sung Cheol Koo ◽

Yurry Um ◽

...

Keyword(s):

Next Generation Sequencing ◽

Simple Sequence Repeat ◽

Next Generation ◽

Sequence Repeat ◽

Codonopsis Lanceolata ◽

Simple Sequence Repeat Markers ◽

Genome Wide ◽

Generation Sequencing ◽

Simple Sequence

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Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing

10.21203/rs.2.12741/v2 ◽

2020 ◽

Author(s):

Hao Bai ◽

Yanghua He ◽

Yi Ding ◽

Huanmin Zhang ◽

Jilan Chen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number ◽

Marek's Disease ◽

Neoplastic Disease ◽

Marek’S Disease ◽

Next Generation ◽

Qrt Pcr ◽

Genome Wide ◽

A Genome ◽

Generation Sequencing

Abstract Background: Marek’s disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD.Results: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1,649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1,360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using qRT-PCR, all of which were successfully confirmed. Finally, qRT-PCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study.Conclusions: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

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Repli-seq: genome-wide analysis of replication timing by next-generation sequencing

10.1101/104653 ◽

2017 ◽

Cited By ~ 8

Author(s):

Claire Marchal ◽

Takayo Sasaki ◽

Daniel Vera ◽

Korey Wilson ◽

Jiao Sima ◽

...

Keyword(s):

Next Generation Sequencing ◽

Replication Timing ◽

Nucleotide Polymorphisms ◽

Robust Methods ◽

Next Generation ◽

Single Nucleotide ◽

Cellular Processes ◽

Genome Wide ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

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Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair derived artifacts as residual errors

10.1101/2019.12.22.886440 ◽

2019 ◽

Author(s):

Xinyue You ◽

Suresh Thiruppathi ◽

Weiying Liu ◽

Yiyi Cao ◽

Mikihiko Naito ◽

...

Keyword(s):

Next Generation Sequencing ◽

Low Frequency ◽

Repair Process ◽

Sequencing Error ◽

Base Substitution ◽

Next Generation ◽

Background Error ◽

Technical Errors ◽

Genome Wide ◽

Generation Sequencing

ABSTRACTTo improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strands-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 6 bp of the library fragments in the 5’-NpCpA-3’ or 5’-NpCpT-3’ trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 6 bp, PECC-Seq could reduce the sequencing error frequency to mid-10−7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10−8. In mutagen-treated TK6, slight increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights in further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

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