425. Detection of microorganisms and mutations by semi-conductor based sequencing
Session: Poster Abstract Session: Novel Devices and Technologies
Thursday, October 18, 2012
Room: SDCC Poster Hall F-H
Background:  Next generation sequencing systems using semi-conductor based detection are very rapid and have very high throughput with bar-coding capabilities. These systems can be used for multiple applications and including identification, detecting of virulence genes, resistance determination and strain typing for novel variants.

Methods: Total nucleic acid was isolated from bacterial and fungal colonies, cell culture media with contaminating pathogens, blood and environmental samples using magnetic beads coated with dextran or silica. For targeted detection of organisms PCR amplification was performed to amplify regions with unique signatures/features. For metagenomic applications of microorganisms bacterial 16S, fungal ITS and/or D2 regions were amplified by PCR. Panels containing 15- 40 organisms (virus, bacteria and fungi) were created by mixing with mammalian cell lines for detection. DNA from cultures was used for WGS to characterize new variants.

Results: We were able to demonstrate a 100% concordance in detection between semi-conductor sequencing and methods like Sanger sequencing, qPCR or PCR-OLA detection. We were able to bar-code 15 samples and get a complete identification of the entire set by sequencing bacterial 16S and the fungal ITS/D2 regions. We were also able to detect resistance conferring mutations in the UL97 region of CMV. Metagenomic samples were also amplified across the entire 16S region and family level identification obtained. The method also used for rapid characterization of the German Enterohemorrhagic Escherichia coli O104:H4 outbreak strain.

Conclusion: Semi-conductor based sequencing methods coupled with targeted PCR amplification have the ability to accurately identify large panels of organisms with high sensitivity. The method can simultaneously detect organisms, associated virulence factors and resistance conferring mutations. Metagenomic analysis can also be enabled on the platform. The high capacity of the system allows bar-coding of multiple samples enabling cost effective operation. In conclusion, semi-conductor based sequencing can provide a single solution for bacterial identification, detection of virulence factors, detection of resistance conferring mutations as well as strain typing.

Lavorka Degoricija, Ph.D.1, Craig Cummings1, Nisha Mulaken, Ph.D.1, Elena Bolchakova, Ph.D.1, jen-Kuei Liu1, Rixun Fang1 and Manohar Furtado, Ph.D.2, (1)Life Technologies, Foster City, CA, (2)Molecular Medicine, Life Technologies, San Ramon, CA


L. Degoricija, Life Technologies: Employee and Shareholder, Salary

C. Cummings, Life Technologies: Employee and Shareholder, Salary

N. Mulaken, Life Technologies: Employee and Shareholder, Salary

E. Bolchakova, Life Technologies: Employee and Shareholder, Salary

J. K. Liu, Life Technologies: Employee and Shareholder, Salary

R. Fang, Life Technologies: Employee and Shareholder, Salary

M. Furtado, Life Technologies: Employee and Shareholder, Salary

Findings in the abstracts are embargoed until 12:01 a.m. PST, Oct. 17th with the exception of research findings presented at the IDWeek press conferences.