nist microbial genomic rms

NIST Microbial Genomic Reference Materials March 18th 2015

Characterization of Bacterial Genomic Reference Materials

Nathan D. OlsonNIST Microbial Research Symposium

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


Why Bacterial Genomic RMs?

Background Material Selection Characterization


Regulatory Needs Quality Control


Selection of NIST Bacterial Genomic RMs



Material Selection and Acquisition

Strain Selection



● For each strain ○ single large batch of DNA ○ ~ 1500 vials

● Produced by local vendor

RM Material


Characterization ofNIST Bacterial Genomic RMs



● Genome Assembly● Base Level Analysis● Genomic Purity● DNA Stability

Characterized Properties



Genome Sequencing

Process

Modified From Loman et al. 2012 Nature Reviews Microbiology 10(9)





Overall structure genome structure



Genome Assembly

Chin et al. 2013

http://www.nature.com/nmeth/journal/v10/n6/full/nmeth.2474.html





Genome AssemblyValidate with Optical Mapping

Mendelowitz and Pop GigaScience 2014

http://www.gigasciencejournal.com/content/3/1/33




Results MG001Identification of a 1.6 Mb Inversion

Optical Mapping Validation





● Purity○ similarity of nucleotides within

a lot

● Homogeneity○ similarity of nucleotides

between vials



Base Level Analysis



Base Level Analysis Homogeneity Results MG001





Presence of DNA from biological contaminants



Genomic Puritytaxonomic read assignment

Hong et al. 2014 Microbiome





Genomic Purity Results MG001Total Contaminants less than 0.005%

Contaminant read count by Taxonomic IDProportion Contaminants





Homogeneity and Stability of DNA fragment size

distribution



DNA Stability



DNA Stability MG001 Results



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.


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.