Skip to main content
SpringerLink
Log in

Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes

  • Protocol
  • First Online:
Adeno-Associated Virus Vectors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1950))

Abstract

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Food and Drug Administration (FDA) (2012) Vaccines and related biological products advisory committee meeting: Cell lines derived from human tumors for vaccine manufacture. FDA briefing Document

    Google Scholar 

  2. Ayuso E, Blouin V, Lock M, McGorray S, Leon X, Alvira MR, Auricchio A, Bucher S, Chtarto A, Clark KR, Darmon C, Doria M, Fountain W, Gao G, Gao K, Giacca M, Kleinschmidt J, Leuchs B, Melas C, Mizukami H, Müller M, Noordman Y, Bockstael O, Ozawa K, Pythoud C, Sumaroka M, Surosky R, Tenenbaum L, van der Linden I, Weins B, Wright JF, Zhang X, Zentilin L, Bosch F, Snyder RO, Moullier P (2014) Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther 25(11):977–987. https://doi.org/10.1089/hum.2014.057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lecomte E, Tournaire B, Cogné B, Dupont J-B, Lindenbaum P, Martin-Fontaine M, Broucque F, Robin C, Hebben M, Merten O-W, Blouin V, François A, Redon R, Moullier P, Léger A (2015) Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing. Mol Ther Nucleic Acids 4:e260. https://doi.org/10.1038/mtna.2015.32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Penaud-Budloo M, Lecomte E, Guy-Duché A, Saleun S, Roulet A, Lopez-Roques C, Tournaire B, Cogné B, Léger A, Blouin V, Lindenbaum P, Moullier P, Ayuso E (2017) Accurate identification and quantification of DNA species by next-generation sequencing in adeno-associated viral vectors produced in insect cells. Hum Gene Ther Methods 28(3):148–162. https://doi.org/10.1089/hgtb.2016.185

    Article  CAS  PubMed  Google Scholar 

  5. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. D’Costa S, Blouin V, Broucque F, Penaud-Budloo M, François A, Perez IC, Le Bec C, Moullier P, Snyder RO, Ayuso E (2016) Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev 5:16019. https://doi.org/10.1038/mtm.2016.19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kozarewa I, Turner DJ (2011) Amplification-free library preparation for paired-end Illumina sequencing. Methods Mol Biol 733:257–266. https://doi.org/10.1007/978-1-61779-089-8_18

    Article  CAS  PubMed  Google Scholar 

  8. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Louis N, Evelegh C, Graham FL (1997) Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology 233(2):423–429. https://doi.org/10.1006/viro.1997.8597

    Article  CAS  PubMed  Google Scholar 

  10. Nandakumar S, Ma H, Khan AS (2017) Whole-genome sequence of the Spodoptera frugiperdaSf9 insect cell line. Genome Announc 5(34). https://doi.org/10.1128/genomeA.00829-17

  11. Kakumani PK, Malhotra P, Mukherjee SK, Bhatnagar RK (2014) A draft genome assembly of the army worm, Spodoptera frugiperda. Genomics 104(2):134–143. https://doi.org/10.1016/j.ygeno.2014.06.005

    Article  CAS  PubMed  Google Scholar 

  12. Penaud-Budloo M, François A, Clément N, Ayuso E (2018) Pharmacology of recombinant adeno-associated virus production. Mol Ther Methods Clin Dev 8:166–180. https://doi.org/10.1016/j.omtm.2018.01.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kondratov O, Marsic D, Crosson SM, Mendez-Gomez HR, Moskalenko O, Mietzsch M, Heilbronn R, Allison JR, Green KB, Agbandje-McKenna M, Zolotukhin S (2017) Direct head-to-head evaluation of recombinant adeno-associated viral vectors manufactured in human versus insect cells. Mol Ther 25(12):2661–2675. https://doi.org/10.1016/j.ymthe.2017.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Magalie Penaud-Budloo and Eduard Ayuso contributed equally to this work.

Authors and Affiliations

  1. INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire de Nantes, Nantes, France

    Emilie Lecomte, Adrien Leger, Magalie Penaud-Budloo & Eduard Ayuso

  2. European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), Cambridge, UK

    Adrien Leger

Authors
  1. Emilie Lecomte
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adrien Leger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Magalie Penaud-Budloo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eduard Ayuso
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eduard Ayuso .

Editor information

Editors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA

    Michael J. Castle

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lecomte, E., Leger, A., Penaud-Budloo, M., Ayuso, E. (2019). Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes. In: Castle, M. (eds) Adeno-Associated Virus Vectors. Methods in Molecular Biology, vol 1950. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9139-6_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9139-6_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9138-9

  • Online ISBN: 978-1-4939-9139-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation