VT-ENSIMAG Biosecurity @ IGEM 2010 Road Map I DNA Synthesis and - - PowerPoint PPT Presentation

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VT-ENSIMAG Biosecurity @ IGEM 2010 Road Map I DNA Synthesis and - - PowerPoint PPT Presentation

Aug 09, 2023 456 likes •867 views

VT-ENSIMAG Biosecurity @ IGEM 2010 Road Map I DNA Synthesis and the Industry II GenoTHREAT Algorithm III An Efficient Software IV Contribution to iGEM V Conclusions and the Road Ahead 2 How is gene synthesis evolving?

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VT-ENSIMAG Biosecurity @ IGEM 2010

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Road Map

I – DNA Synthesis and the Industry II – GenoTHREAT Algorithm III – An Efficient Software IV – Contribution to iGEM V – Conclusions and the Road Ahead

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How is gene synthesis evolving? Why is it important?

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The Guardian

  • Ordered portion of Small Pox Sequence (2006)

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1918 Flu Epidemic

  • Killed over 35,000,000 people worldwide
  • Reconstructed in 2005 at the CDC

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Mycoplasma Mycoides

  • Synthetic genome completed in

2010

  • Functional and self-replicating
  • Huge positive impact in the

biological and medicinal field.

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What relates the three controversial events?

De novo gene synthesis

What might the future hold?

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Industry Response to Dual Use

  • Vague
  • Implementation left

to Companies

  • Do not apply to all

companies

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U.S Government Guidance

  • “Screening Framework Guidance for

Providers of Synthetic Double Stranded DNA”

  • Draft published in November 2009
  • Final version published in October 2010

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Where do we fit in? GenoTHREAT is the first implementation of the sequence screening guidance

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Road Map

I – Danger of DNA synthesis II – GenoTHREAT algorithm III – An efficient software IV – Contribution to iGEM V – Conclusion

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GenoTHREAT algorithm

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What should we do with subsequences?

Even one subsequence flagged Every subsequence passed 13

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What constitutes a dangerous sequence?

Gene derived from, or encoding for, a Select Agent or Toxin

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Best matches Mus musculus Mus musculus BLAST results Score Mus musculus 100 Mus musculus 100 Danio rerio 97 Danio rerio 43

BLAST

Is this subsequence a hit?

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Best matches Lumpy skin disease virus Sheeppox virus BLAST results Score Lumpy skin disease virus 100 Sheeppox virus 100 Goatpox virus 98 Dearpox virus 44

BLAST

Is this subsequence a hit?

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Best matches Bacillus anthracis Bacillus cereus BLAST results Score Bacillus anthracis 100 Bacillus cereus 100 Plasmodium falciparum 63 Clostridium ljungdahlii 44

BLAST

Is this subsequence a hit?

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Road Map

I – Danger of DNA Synthesis II – GenoTHREAT Algorithm III – An efficient software

1 – Database of Test Sequences 2 – Testing GenoTHREAT boundaries 3 – Time Efficiency

IV – Contribution to iGEM V – Conclusions and the Road Ahead

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Database of Test Sequences

  • 184 sequences manually selected straight from

GenBank

  • Contains both dangerous and harmless

sequences

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Keyword List Content Variation

  • Limited keyword list:

uniquely composed of words in SAT List

  • Extensive keyword list:

extension of limited keyword list containing words uniquely related to SAT.

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Keyword list method not mentioned in guidance

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Database of Test Sequences

  • Database of Modified sequences
  • Created by recombining Unmodified sequences

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Road Map

I – Danger of DNA Synthesis II – GenoTHREAT Algorithm III – An efficient software

1 – Database of Test Sequences 2 – Testing GenoTHREAT boundaries 3 – Time Efficiency

IV – Contribution to iGEM V – Conclusions and the Road Ahead

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Intervening Sequences

Potential Danger: SAT sequences hidden within larger, benign sequences Conclusion: All hidden sequences were detected

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Draft Guidance sets screening limit at 200bps/66aa

300bps NSAT 200bps SAT 300bps NSAT 300bps NSAT 300bps NSAT 250bps SAT

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Degenerate Sequences

Potential Danger: Codon optimized nucleotide sequences

GATTTGGACACTCATTTCACC DLDTHFT Unmodified Nucleotide Degenerate Nucleotide GATACGTCAACCTTTTAAGC Amino Acid Sequence

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Degenerate Sequences

  • 100% of hits were correct
  • All due to amino acid subsequence

Conclusion: Necessity of the Six Frame Translation

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Road Map

I – Danger of DNA Synthesis II – GenoTHREAT Algorithm III – An efficient software

1 – Database of Test Sequences 2 – Testing GenoTHREAT boundaries 3 – Time Efficiency

IV – Contribution to iGEM V – Conclusions and the Road Ahead

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Screening time & hardware

27 Online Desktop Business Class Server

Sequence length (bps) Screening time (min)* 2000 2 10,000 12.5

*Screening performed using business class server

Conclusion: Viable at 100’s

  • f sequences screened a day
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Road Map

I – Danger of DNA synthesis II – GenoTHREAT algorithm III – An efficient software IV – Contribution to iGEM V – Conclusions and the Road Ahead

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The iGEM Registry

Why screening the registry? Results:

Initial Screen: Sequence 1 – 1724

  • Hit Rate: 6.5%

Modified Program and Parameters

  • Hit Rate: 2.9%

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Conclusion: Potentially viable in large scale applications

  • Real world gene order

simulation

  • Contribution to iGEM
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Legitimate threat identified

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Road Map

I – Danger of DNA synthesis II – GenoTHREAT algorithm III – An efficient software IV – Contribution to iGEM V – Conclusions and the Road Ahead

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Conclusions

  • Screening quality dependent upon implementations
  • Developed test set for future research
  • Draft Guidance provides good framework for sequence

screening software

  • Vague on certain points
  • Multiple possible implementation of varying quality
  • GenoTHREAT is a viable software:
  • Reasonable time
  • Accurate detection of sequences of concern
  • Decent numbers of false positives (human review)

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And iGEM!

GenoTHREAT and the Registry:

  • Provide a safer working environment for students

and the public

  • Foster development of responsible scientists

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And iGEM!

iGEM Collaboration:

  • Developed software to design

primers

  • USER Fusion is standard for scarless

restriction

  • Virginia United iGEM Team
  • Primer design is time consuming

and error prone

  • Very useful for other teams in the

future

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The Road Ahead

Source: http://www.sepiamutiny.com/sepia/archives/cat_law.html

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The Road Ahead

Source: http://www.ccmr.cornell.edu/igert/

NSF Award for $100,000 to continue work

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The Road Ahead

Source: http://www.globalsecurity.org/wmd/intro/bio_ricin-pics.htm and http://www.bbc.co.uk/drama/smallpox2002/ and http://frameshiftcoaching.wordpress.com/2009/12/

GenoTHREAT

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VT-ENSIMAG biosecurity

Team:

  • Gaëlle Letort
  • Michael Kozar
  • Olivier Mirat
  • Arunima Srivastava
  • Tyler Stewart

Advisors:

  • Dr. Jean Peccoud
  • Laura Adam

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Questions?