Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
TOWARDS DEVELOPMENT OF A STANDARD INPUT
FOR DETECTION OF MICROBES BY NEXT
GENERATION SEQUENCING
Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.02)
Rachel Spurbeck, Richard Chou, Nick Fackler, Jazmine Quinn
Battelle Memorial Institute
The utilization of Next Generation Sequencing (NGS) for bacterial pathogen detection is a powerful
technique for biosurveillance purposes. However, the field suffers from a lack of standards for
validation of NGS pipelines necessary to determine the limit of detection. Currently, there are several
different methods for DNA extraction, library preparation, sequencing, and bioinformatics from
which a researcher can pick and choose, without a means for determining which is best for their application.
Presented here is the initial development of a standard for microbial detection by NGS. This
standard is a quantified mixture of bacterial cells, which when sequenced using our chosen method,
produce an expected number of reads mapping to the genomes of the bacteria present in the standard
mix. To develop this standard, pure cultures of Staphylococcus aureus, Pseudomonas aeruginosa,
and Escherichia coli were quantified by spectrophotometry (A600) and spread plates. Pure cultures
were then diluted in a titration series from 1010 CFU to 102 CFU, DNA was extracted, and
PCR‐free libraries were prepared. Prior to quantification and sequencing on the Illumina MiSeq,
the libraries were pooled and concentrated. Similarly, two organism mixtures (P. aeruginosa + E.
coli, P. aeruginosa + S. aureus, E. coli + P. aeruginosa, and S. aureus + P. aeruginosa) were prepared
from the quantified pure cultures with one organism being held constant at 108 CFU and
the other organism in a titration series from 108 to 102 CFU. DNA was then extracted from the
mixtures, PCR‐free libraries prepared, and sequenced. The number of reads that mapped to each
genome in the pure culture library titrations or in the mixed samples were quantified. For each of
the bacteria in pure culture, read counts plateaued near 105 reads between 106 and 102 CFU. Thus,
for individual or low DNA samples, each bacteria was detectable, but not quantifiable below 106
CFU. For TSB negative culture control, read counts were under 100 reads mapping to any of the
queried genomes. For mixed cultures, where there was more background DNA to enable efficient
reactions during library preparation, the plateau effect below 106 was not observed, consistent with
the hypothesis that the presence of a threshold of DNA is necessary for efficient library preparation.
With a background present, the coefficient of determination (R2) is 0.9667 with a P‐value of 0.000421
for a titration of E. coli in a P. aeruginosa background, demonstrating that there is a correlation
between the number of bacteria in a sample and the number of reads mapping to that organism.
Future work is necessary to develop the standard further, by including more organisms, and testing
the mix in different backgrounds. Using a known mix of organisms in a known quantity will enable
true comparison and evaluation of bacterial detection systems and workflows, which is necessary for
the general acceptance of pathogen detection results.
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