Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

Suckling, Lorna; McFarlane, C.R.; Sawyer, Chelsea; Chambers, Stephen P.; Kitney, R.I.; McClymont, David; Freemont, Paul S.

doi:10.1016/j.synbio.2019.01.002

Cited by 18 publications

(13 citation statements)

References 17 publications

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“…The tagmentation library preparation method has been shown to work for circular plasmid DNA and, in addition to VGS, is used to sequence verify the plasmids used for AAV production. 21 After amplification, the library is normalized, sequenced and the data are provided to Addgene for analysis ( Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

et al. 2020

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Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as doublestranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.

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Section: Resultsmentioning

confidence: 99%

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

et al. 2020

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“…DNA was used to prepare NGS library using Illumina ® Nextera XT DNA Library Preparation Kit (FC-131-1096, Illumina Inc., CA, USA) and Nextera XT Index Kit v2 Set A (FC-131-2001, Illumina Inc., CA, USA) by following manufacturer's instructions [60]. The generated DNA libraries were pooled and loaded…”

Section: Ngs Library Preparationmentioning

confidence: 99%

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

Ok¹,

J²,

Chang³

2020

Preprint

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BackgroundMicrobiota plays an important role in food safety and its alteration poses a serious threat to humans. Comparative microbiome profiling using next-generation sequencing (NGS) enabled the understanding of microbial diversity and similarity between different species. In this study, we used NGS to profile the fecal microbiota of sick human and broiler chickens. A total of 26 fecal samples were collected from severely sick human subjects (n= 13) and broiler chickens (n=13) with similar symptoms.ResultsThe total number of microbial species detected in broiler chickens fecal microbiota was higher than that of humans. Phylum Proteobacteria was the most abundant in both human and broiler chickens fecal microbiota while Tenericutes was found to be least abundant in both species. Phylum Actinobacteria was found only in the human fecal microbiota. In both humans and broiler chickens, E.coli was found to be phylogenetically related suggesting a microbial association between both species.ConclusionNGS based taxonomic profiling revealed the association of microbial dysbiosis with extreme sickness in both humans and broiler chickens. The dominance of phylum Proteobacteria in both the species ascertains their altered gut microbiota. Both human and broiler chickens microbial communities were found to be genetically related indicating horizontal transfer of microbes between the two species.

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Section: Ngs Library Preparationmentioning

confidence: 99%

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

Afridi

J²,

Chang

2020

Preprint

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Background: Microbiota plays an important role in food safety and its alteration poses a serious threat to humans. Comparative microbiome profiling using next-generation sequencing (NGS) enabled the understanding of microbial diversity and similarity between different species. In this study, we used NGS to profile the fecal microbiota of sick human and broiler chickens. A total of 26 fecal samples were collected from severely sick human subjects (n= 13) and broiler chickens (n=13) with similar symptoms. Results: The total number of microbial species detected in broiler chickens fecal microbiota was higher than that of humans. Phylum Proteobacteria was the most abundant in both human and broiler chickens fecal microbiota while Tenericutes was found to be least abundant in both species. Phylum Actinobacteria was found only in the human fecal microbiota. In both humans and broiler chickens, E.coli was found to be phylogenetically related suggesting a microbial association between both species. Conclusion: NGS based taxonomic profiling revealed the association of microbial dysbiosis with extreme sickness in both humans and broiler chickens. The dominance of phylum Proteobacteria in both the species ascertains their altered gut microbiota. Both human and broiler chickens microbial communities were found to be genetically related indicating horizontal transfer of microbes between the two species.

show abstract

Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

Cited by 18 publications

References 17 publications

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

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