whole genome sequencing analysis for top seven shiga toxin … · 2019. 7. 31. · whole genome...
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RESULTS (Continued)
Table 1. Comparison of wgMLST and wgSNP
wgMLST Allelic Differences
wgSNP Analysis with
Strict Filtering(Ref Genome: E coli O157:H7 str.
Sakai: NCBI - NC_002695.2)
METHODS (Continued)DNA Extraction and Sequencing
Data Analysis
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July 2019
INTRODUCTION Escherichia coli O157:H7 and six other Shiga
toxin-producing E. coli (STEC) serogroups O26, O45,
O103, O111, O121, and O145 are often referred to as
the Top 7 STEC.
Subtyping of STEC is important for outbreak
investigation. PFGE, MLVA, and rep-PCR are some
of the traditional investigation methods but they are
either time consuming or of low resolution.
Whole genome sequencing (WGS) technology has
been widely applied to speciation, subtyping, and
distinguishing closely related strains. The
performance of WGS must be examined to ensure
appropriate implementation for STEC.
Whole Genome Sequencing Analysis for Top Seven Shiga Toxin-producing Escherichia coli
Authors: Jiaojie Zheng1, Sarita Raengpradub
Wheeler1, Xuwen Wieneke1, Timothy Freier1
1Mérieux NutriSciences, Chicago, Illinois, USA
International Association for Food Protection
Annual Meeting
July 21-24, 2019 | Louisville, KY
OBJECTIVETo assess the impact of selecting wgMLST or
wgSNP pipelines on differentiation of Top 7 STEC
strains
RESULTSSequence Data Quality All samples except sample ET18 passed quality
control. ET18, which had low average genome
coverage (15), was omitted and not analyzed further.
wgMLST and wgSNP Analyses The wgMLST similarity coefficient dendrogram
(Figure 1) shows the relationship of 30 STEC samples
and 5 NCBI genomes (similarity percentage showed
on the nodes)
• Within the Top 7 STEC groups, any two strains
with different serotypes shared no more than
88.1% similarity.
• O157:H7 strains were well distinguished from
other non-O157: H7 strains with only 9.7%
similarity to other STEC.
• Six O157:H7 strains shared at least 98.2%
similarity. Two groups of two O121 strains had over
98% similarity.
Table 1 shows the comparison of wgMLST and
wgSNP analyzing strains with the same serotype.
• Both wgMLST and wgSNP were able to further
differentiate strains with the same serotype.
• The results from two methods agreed to each
other, despite the numeric differences.
• One difference between the two methods is
wgMLST is only available for genus with
established database while wgSNP requires a
reference genome.
Isolate Preparation:
Cultured confirmed
isolates on TSA
DNA Extraction:
Qiagen DNeasy
UltraClean microbial kit
Library Preparation:
Illumina Nextera XT
library preparation kit
Sequencing:
Illumina MiSeq
sequencer
CONCLUSIONS wgMLST similarity coefficient clustering (showing
percent similarity) shows the relatedness among all
strains and is able to separate Top 7 STEC strains
belonging to different serotypes without the need of a
reference genome.
For strains of the same serotype, both wgMLST
(showing allelic differences) and wgSNP (showing
number of SNPs) analyses can be used to further
identify genomic differences between strains.
Sequence Data Quality Control
● Checked raw data quality using BioNumerics software
● Genome coverage: > 30
● Download NCBI genome sequence data to compare
E. coli O26: SRR7827094
E. coli O103: SRR7828044
E. coli O111: SRR7827093
E. coli O121: SRR5816151
E. coli O145: SRR7828323
wgMLST
● Analyzed using BioNumerics software
● De novo assembly
● Assembly based calls
● Assembly free calls
● wgMLST clustering (UPGMA method)
● Download NCBI complete genome as
reference
E. coli O157:H7 str. Sakai: NC_002695.2
wgSNP
● Analyzed using BioNumerics software
● Used NCBI complete genome as
reference
● SNP mapping to reference genome
● wgSNP clustering (UPGMA method)
METHODSIsolates A total of 30 E. coli isolates were sequenced and
analyzed in this study. The number of isolates for
each serogroup are listed below.
O26 O45 O103 O111 O121 O145O157:
H7
Other
O157
No. of isolates
4 4 4 4 4 1 6 3
Figure 1. wgMLST similarity coefficient dendrogram
Whole Genome Sequencing Analysis for Top Seven Shiga Toxin-producing Escherichia coli
Poster
International Association for Food Protection Annual Meeting
Louisville, KY July 21 - 24, 2019
MERIEUX NUTRISCIENCES