Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
COMPARISON OF STANDARD CULTURE AND
SINGLE-COLONY DNA EXTRACTION METHODS FOR
WHOLE-GENOME SEQUENCING OF
CAMPYLOBACTER JEJUNI
Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.12)
Kun Liu, Karen Jinneman
US FDA
Introduction
Campylobacter jejuni is the most common food‐borne bacteria causing diarrhea in the US and is most often associated with
poultry, raw milk, unpasteurized eggs, contaminated water or produce. The technical advancement and decrease in cost of
whole‐genome sequencing (WGS) have made WGS more widely available for rapid and accurate identification of
pathogens such as C. jejuni from contaminated food. Since bacterial culture and DNA extraction are critical to WGS, we
evaluated the effects of two available methods on WGS data quality and analysis.
Purpose
To compare the quality of C. jejuni genomes prepared from two extraction methods. The subculture method, currently
used in FDA, requires more biomass and involves an automated extraction. In contrast, a more rapid protocol needs only
a single colony and mechanically releases DNA, which shows potential to greatly reduce culture time and reagent cost.
Here, DNA was prepared from both methods, data quality was compared and method applications were evaluated based
on WGS results.
Methods
C. jejuni B9 strain was isolated by Pacific Laboratory Northwest in 1983. DNA was extracted using QIAcube (Qiagen Inc.,
Valencia, CA) from subculture 1 or single‐colony 2. DNA concentrations were determined by Qubit HS kit (Thermo Fisher
Scientific Corp., Waltham, MA). Libraries were prepared with Nextera XT Kit (Illumina Inc., San Diego, CA). WGS
experiments were performed on MiSeq (Illumina Inc., San Diego CA). Genomes were assembled with CLC genomics
workbench (Qiagen Inc.). WGS data were evaluated using SpeciesFinder for 16S typing 3 and KmerFinder for speciation 3,
4. Genome annotations were performed with Rapid Annotation using Subsystem Technology (RAST) 5‐7.
Results
The subculture method took 48 hours to grow C. jejuni and 2 hours on extraction, whereas the single‐colony method took
less than 30 minutes to finish all steps. The former yielded higher DNA concentration than the latter (11.4 vs. 0.5 ng/ul),
but both were sufficient for library preparation which only requires 0.2 ng/ul.
Qualitatively, B9 was correctly identified to be C. jejuni based on WGS data from both extraction methods and highly
homologous to reference strains NCTC‐11168 and RM‐1221. Quantitatively, de novo assembly from the subculture
method yielded an N50 length of 188 kb from 63 contigs. In comparison, that from the single‐colony method yielded an
N50 length of 72 kb from 244 contigs. Average coverage rates were both greater than 270x. RAST annotations showed
similar results. Thedraft genome from subculture was predicted to contain 1751 coding sequences, 315 subsystems, and
43 RNAs, while that from single‐colony was predicted with 1753 coding sequences, 312 subsystems, and 43 RNAs.
Conclusions
We compared two DNA preparation methods on a C. jejuni field isolate and evaluated WGS data generated to assess
applications for regulatory food safety science. For qualitative analysis following WGS, such as confirmation and speciation,
the single‐colony method is as good as subculture with a great time‐saving advantage (49.5 hours shorter) and reduced
cost ($4.60 less per sample). However, for deeper genomics analysis, DNA from subculture exhibits better quality based on
N50 and contig counts.
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