Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
ASSESSING THE SINGLE NUCLEOTIDE
POLYMORPHISM (SNP) VARIABILITY OF OUTBREAK
STRAINS DURING IN VITRO PASSAGE
Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.03)
Ashley Sabol
Centers for Disease Control and Prevention
Foodborne bacterial pathogens are responsible for approximately 9.4 million illnesses each year.
The mode in which these organisms are passed from environmental or food source to humans can be
varied. Furthermore, the rate at which these pathogens change can be identified through molecular
subtyping of strains that belong to the same outbreak or source or by passaging them several times
in vitro or in vivo. It is important to gain insight on the observed strain variability that may occur
due to laboratory manipulations such as culture passages especially after recovery from a patient
– in order to accurately distinguish genetic differences (unrelated strains) from background variation
resulting from laboratory manipulations. This is particularly important for high resolution methods,
such as whole genome sequencing (WGS) which is currently being adopted by the US public health
laboratories for routine surveillance and outbreak investigations of foodborne bacterial pathogens.
Four outbreak‐associated strains across 4 species Salmonella enterica serovar Newport, Shiga toxinproducing
Escherichia coli (STEC) serogroup O157:H7, Vibrio cholerae, and Listeria monocytogenes
– were passed 20 times in vitro. Three colony picks were selected at passes 1, 5, 10, 15, & 20 and
streaked to blood agar plates. After incubation for 16‐24 hours at 37°C, DNA extractions were
performed for each pick, DNA libraries were prepared using the NexteraXT kit (Illumina Inc.) and
sequenced paired‐end on the Illumina MiSeq (500 cycles). The high quality single nucleotide polymorphism
(hqSNP) calls were determined using Lyve‐Set v1.1.4e (http://github.com/lskatz/Lyve‐SET)
at 10x coverage, 75% frequency for Listeria and Vibrio, and 20x coverage, 95% frequency for STEC
and Salmonella. SNPs clustered closer than 5 bp were filtered.
Over the course of 20 passes, anywhere from 0‐6 hqSNPs were observed among the 15 colony picks
sequenced for each strain. The higher number of hqSNP differences for each strain were observed
among picks from the later passes (i.e. passes 10‐20). The least number of SNP differences between
picks were observed among the STEC colony picks with 0‐1 SNPs observed. The most SNP differences
for a single pick were identified for Listeria pass 15, where 4‐6 SNP differences were counted
when compared to all other picks for that strain.
This study has provided preliminary evidence that WGS data, as measured by hqSNP analysis, remains
stable despite of in vitro manipulations and can hence be used to assess genetic relatedness among
foodborne bacterial strains during outbreak investigations. Future plans for this study are to expand
it to include Campylobacter spp. and an additional Salmonella enterica serotype, as well as exploring
different analysis approaches such as whole genome multi‐locus sequence typing (wgMLST).
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