Sequencing
SFAF2016%20Meeting%20Guide%20Final%203
SFAF2016%20Meeting%20Guide%20Final%203
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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting<br />
DISTINGUISHING OUTBREAKS OF BOTULISM USING<br />
A REFERENCE-BASED SNP ANALYSIS AND HIGH<br />
QUALITY REFERENCE GENOME SEQUENCES OF<br />
CLOSTRIDIUM BOTULINUM<br />
Thursday, 2nd June 11:20 La Fonda Ballroom Talk (OS‐5.03)<br />
Brian Shirey 1 , Shannon Johnson 2 , Maliha Ishaq 1 , Carolina Luquez 1 ,<br />
Karen Hill 2 , Susan Maslanka 1<br />
1 Centers for Disease Control and Prevention, 2 Los Alamos National Laboratory<br />
Reference‐based whole genome SNP analysis can be used to support laboratory investigations of<br />
botulism by providing the necessary resolution, speed, and accuracy to distinguish outbreakassociated<br />
strains from sporadic cases. The National Botulism Laboratory Team (NBLT) at CDC has<br />
partnered with Los Alamos National Laboratory (LANL) to generate additional high‐quality, finished<br />
Clostridium spp. reference sequences, and develop a user‐friendly, reproducible SNP‐based workflow<br />
that utilizes open‐source, freely‐accessible bioinformatics tools.<br />
Currently, there are 20 complete genome sequences of botulinum neurotoxin producing clostridia<br />
(BTPC) available in the NCBI database; however, many commonly‐occurring BTPC strains lack<br />
representation in the database. These gaps in representation limit our ability to perform highresolution,<br />
reference‐based BTPC strain subtyping. The Clostridium botulinum PulseNet database of<br />
PFGE patterns was used, in part, to identify 10 commonly‐occurring BTPC strains that lack sequence<br />
representation in public databases. The LANL Genomics Group sequenced (using PacBio and Illumina<br />
platforms), assembled, and annotated the genomes of these 10 strains to generate high‐quality,<br />
complete genome sequences that will be publically available in the NCBI database.<br />
NBLT used the Ion Torrent PGM to sequence an additional 100 BTPC strains to facilitate the evaluation of<br />
open‐source bioinformatics tools for reference‐based SNP genotyping. Through this effort, NBLT has<br />
successfully identified an analytical workflow to: 1) rapidly identify the most appropriate BTPC reference<br />
sequences based on average nucleotide identity thresholds, 2) construct accurate SNP phylogenies using the<br />
RealPhy v112 tool, and 3) identify a BoNT serotype‐specific parameter regime for distinguishing BTPC<br />
strains based on estimates of evolutionary divergence. This method can be employed by users with minimal<br />
bioinformatics expertise to rapidly and accurately distinguish BTPC strains within a single botulism<br />
outbreak, or between a small number of outbreaks, without relying on high performance computing<br />
requirements. This workflow will facilitate labora‐ tory investigations of botulism by providing a<br />
reproducible, yet customizable subtyping tool with proven efficacy to distinguish outbreak strains.<br />
The findings and conclusions in this report are those of the authors and do not necessarily represent<br />
the official position of the Centers for Disease Control and Prevention.<br />
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