E1 - PacBio. E. faeciumST17. E6 - illumina. After assembly of PacBio sequences with RS_HGAP_Assembly.2 [Chin-2013] we get a finished E1 genome with one ...
S e q u e n c in g , a ss e m b ly a n d c o m p a ra tiv e g e n o m ic s o f six E n te r o c o c c u s fa e c iu m S T 1 1 7 , an emergent multiresistant clone responsible for an increase of bacteremia and fecal carriage in Spain 1
1
1
1
R. Tobes , M. Manrique , P. Pareja-Tobes , E. Pareja-Tobes , E. Pareja
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1. Oh no sequences! Research group. Era7 Bioinformatics. Madrid. Spain 2, 3, 4
A.P. Tedim
2, 3, 5
, P. Ruiz-Garbajosa
, R. Cantón
2, 3, 5
, F. Baquero
2, 3, 4
, T. Coque
2, 3, 4
2. Department of Microbiology, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 3. Unidad de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain; 4. Centros de Investigación Biomédica en Red de Epidemiología y Salud Pública, (CIBER-ESP); 5. Red Española de Investigación en Patología Infecciosa (REIPI)
An abrupt emergence of an AmpR ST117 Enterococcus faecium(Efm) clone associated with
E. faeciumST117 E1- PacBio
E. faeciumST117 E1- illumina
a dramatic increase in the rates of bacteremia and fecal carriage by Efm at different Spanish hospitals since 2009. This clone belongs to the human adapted ST78 Efm lineage. We analyze variation at genome level of phenotypically diverse Efm ST117from patients
E. faeciumST117 E1- PacBio
E. faeciumST117 E2- illumina
attending in Madrid area (2009-2012) and describe the first completed ST117 genome. After assembly of PacBio sequences with RS_HGAP_Assembly.2 [Chin-2013] we get a
E. faeciumST117 E1- PacBio
E. faeciumST117 E3- illumina
finished E1 genome with one chromosome and 5 plasmids (1 MegaPL, 1 medium size PL and 3 small size PL).
PacBio assembly allowed to perfectly define the plasmids, even the small size ones. The
E. faeciumST117 E1- PacBio
E. faeciumST117 E4- illumina
existance and the size of these 5 plasmids was experimentally tested.
Independently we sequenced E1 genome with illumina and did the assembly with velvet. In
E. faecium ST117
the figure we show the pair-wise alignment of the PacBio assembly and the illumina
E1- PacBio
E. faeciumST117 E5- illumina
assembly. The PacBio assembly has a significant larger size. The additional sequence fragments in PacBio assembly (in the figure the white regions in the MAUVE blocks corresponding to the PacBio E1 genome) are mainly trasposases and other mobile
E. faeciumST117 E1- PacBio
E. faeciumST17 E6- illumina
elements and RNA operon copies that are probably colapsed in the illumina velvet assembly. PacBio sequencing is especially useful for defining all the copies of each gene.
Whole genome sequencing (WGS) with PacBio allowed to
COMPARATIVE ANALYSIS
get the firts E. faecium ST117 finished genome. Using PacBio we have been able to solve the elements with
Strain characterization at whole genome level - Resi sta nce genes l ocat ion
CORE GENOME SNPs The most similar genome
- Virulence ge nes location - M obi le Genetic Ele ments (M GE) - Plasmids: Number, Size,
Ma pping reads to the reference genome
Repli cat ion, Conjuga tion, M aintenance modules - Transposons - IS elem ents - Hori zontal tra nsfer de tection
ACCESORY GENOME
Sequences). These repeated MGE elements frequently bears Pair-wise whole genome comparison with Di fferences prog ram: rearrangements, SNPs, Inserti ons and Deletions of any size in the BG7 functional a nnota tion context
SNPs comparison Phylogeneti c trees
many different copies as MGE (transposons, Insertion
Orthologous ta ble Parallel coordinates
virulence and antibiotic resistance genes that is important to solve and analyze. Plasmids are difficult to assembly because they bear a high number of MGE. Thus PacBio is a specially useful technology for working with bacterial plasmids. Our new comparative genomics pipelines allow the detection
Studies to get insight into the dynamic evolution of AbR and the
of differences that are not detected by classical methods.
pathogenicity of this clone in the hospital setting are in progress
The 6 E. faecium genomes with their BG7 annotations will be
(Manuscript in prepration).
publicly available within the next months.
ACKNOWLEDGEMENTS: Work at HRYC-IRYCIS is supported by research grants funded by the European Commission (EvoTAR-282004), the Ministry of Economy and Competitiveness of Spain (NEXT-MICRO-IDI-20120242, FIS-PI12-01581) and the Spanish research networks CIBER-ESP (CB06/02/0053), REIPI RD12/0015), PROMPT (S2010/BMD2414), and REDEEX (BFU 2008-0079-E/BMC).
Work at Era7 Bioinformatics was suppor ted in part by Ministry of Economy and Competitiveness of Spain (NEXT-MICRO-IDI-20120242 and by Era7 that funds
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