Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
EVALUATION OF AVERAGE NUCLEOTIDE IDENTITY
USING MUMMER (ANI-M) AND RPOB GENE
PHYLOGENY FOR IDENTIFICATION OF
VIBRIONACEAE BY WHOLE GENOME SEQUENCE
ANALYSIS
Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.06)
Monica Santovenia 1 , Maryann Turnsek 2 , Lee Katz 2 , Grant Williams 1 ,
Jonathan Jackson 1 , Cheryl Tarr 2
1 IHRC/CDC, 2 Centers for Disease Control and Prevention
A database that integrates multiple methods for analysis of whole genome sequence (WGS) data
for identification of enteric pathogens including Vibrionaceae is being developed at CDC using
BioNumerics v7.5 (Applied Maths) as a software platform. We evaluated for possible inclusion two
methods for species identification: Average Nucleotide Identity using MUMmer (ANI‐m), which
provides a pairwise similarity between two genomes; and rpoB gene phylogeny, which places isolate
sequences into a larger phylogenetic context with other Vibrio species.
ANI‐m and rpoB gene phylogeny were evaluated using >80 Vibrionaceae genome assemblies, representing
15 clinically relevant Vibrio species. Genomic DNA was extracted using the ArchivePureTM
DNA Cell/Tissue Kit (5 PRIMETM), and sequencing was performed on the Illumina MiSeq and Pacific
Biosciences Single Molecule, Real Time (SMRT) sequencer platfor ANI‐m was calculated using inhouse
developed scripts on a high performance computer. The rpoB gene sequences were aligned
and phylogenetic analysis performed in MEGA v5 using the Neighbor‐joining algorithm.
The phylogeny based on rpoB gene‐sequence variation grouped isolates of each species into clusters
that were clearly delineated from other Vibrio species. Overall genome similarity based on ANI‐m
was generally 95% for members within a species for the clinically‐relevant Vibrionaceae. The rpoB
phylogeny provides more information about the affinities of different species (e.g. V. navarrensis is
closely related to V. vulnificus) but requires expertise in interpreting gene trees. ANI‐m provides a
more simplistic estimator for delineating species boundaries, but cannot guide additional testing if
a query sequence does not match any genomes in a comparative database.
These approaches show promise for identification of Vibrionaceae, and further evaluation and validation
on a larger set of genomes is currently ongoing. Also, rMLST, which analyzes variation in the
53 genes encoding the bacterial ribosome protein subunits (rps genes), will be evaluated for possible
integration into the database.
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