nist microbial genomic rms
TRANSCRIPT
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NIST Microbial Genomic Reference Materials March 18th 2015
Characterization of Bacterial Genomic Reference Materials
Nathan D. OlsonNIST Microbial Research Symposium
March 18th, 2015
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NIST Microbial Genomic Reference Materials March 18th 2015
Certain commercial equipment, instruments, or materials are identified in this
presentation in order to specify the experimental procedure adequately. Such
identification is not intended to imply recommendation or endorsement by the
National Institute of Standards and Technology, or affiliated venues nor is it
intended to imply that the materials or equipment identified are necessarily the
best available for the purpose.
All opinions expressed in this presentation are the authors’ and do not necessarily
reflect the policies and views of NIST or affiliated venues.
Disclaimer
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NIST Microbial Genomic Reference Materials March 18th 2015
Why Bacterial Genomic RMs?
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Regulatory Needs Quality Control
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NIST Microbial Genomic Reference Materials March 18th 2015
Selection of NIST Bacterial Genomic RMs
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Material Selection and Acquisition
Strain Selection
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● For each strain ○ single large batch of DNA ○ ~ 1500 vials
● Produced by local vendor
RM Material
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NIST Microbial Genomic Reference Materials March 18th 2015
Characterization ofNIST Bacterial Genomic RMs
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability
Characterized Properties
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Genome Sequencing
Process
Modified From Loman et al. 2012 Nature Reviews Microbiology 10(9)
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability
Characterized Properties
Overall structure genome structure
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Genome Assembly
Chin et al. 2013
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Genome AssemblyValidate with Optical Mapping
Mendelowitz and Pop GigaScience 2014
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Results MG001Identification of a 1.6 Mb Inversion
Optical Mapping Validation
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability
Characterized Properties
● Purity○ similarity of nucleotides within
a lot
● Homogeneity○ similarity of nucleotides
between vials
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Base Level Analysis
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Base Level Analysis Homogeneity Results MG001
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability
Characterized Properties
Presence of DNA from biological contaminants
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Genomic Puritytaxonomic read assignment
Hong et al. 2014 Microbiome
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Genomic Purity Results MG001Total Contaminants less than 0.005%
Contaminant read count by Taxonomic IDProportion Contaminants
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability
Characterized Properties
Homogeneity and Stability of DNA fragment size
distribution
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
DNA Stability
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
DNA Stability MG001 Results
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Background Material Selection Characterization
NIST Microbial Genomic Reference Materials March 18th 2015
Conclusions● Microbial genomic reference materials characterized:
○ Genome Assembly○ Base Level Analysis○ Genomic Purity○ DNA Stability
● Reference material and data will help validate sequencing and bioinformatic processes.
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NIST Microbial Genomic Reference Materials March 18th 2015
Acknowledgements● From FDA:
○ Heike Sichtig○ Marc Allard○ Tim Muruvanda○ Shashi Sharma○ Nagarajan Thirunavukkarasu
● From NIST:○ Marc Salit○ Justin Zook○ Scott Jackson○ Nancy Lin○ Jenny McDaniel○ Lindsay Vang○ David Catoe○ Steven Lund
This work was supported by the Department of Homeland Security (DHS) Science and Technology Directorate under the Interagency Agreement HSHQPM-12-X-00078 with NIST and by two interagency agreements with the FDA.