1. SPECIES AT THE GENOMIC LEVEL

2. DDH has been the gold standard  the “sex” for higher eukaryotes Stackebrandt et al., 2002, Int J Syst Evol Microbiol. 52:846-849 Rosselló-Mora & Amann 2001, FEMS Rev. 25:39-67 Gevers et al., 2005, Nature Rev. Microbiol. 3:733-739 DDH (DNA-DNA hybridization):  70% similarity (50-70%)  used since the 60’s  strong influence  non cumulative DB  need to be substituted MLSA (multilocus sequence analysis):  5-10 full/partial sequences  house keeping genes  primer design difficulties  biases in the selection of genes  time consuming  ↓↓ number for stable topology Amplify and sequence 5-10 housekeeping genes for each strain Concatenate gene sequences Reconstruct the phylogeny genAgenBgenCgenDgenEgenF Str. 1 Str. 2 Str. 3 Str. 4

3. Alternative approaches  ANI Konstantinidis and Tiedje, 2005, PNAS. 102:2567-2572 Genome a BLAST N Genome b Search annotated ORFs sieve common orthologous genes ANI aa b genome Cut into fragments of 1020 nuc + BLAST N < 30% identity < 70% aligned seq > 30% identity > 70% aligned seq discard ANI Goris et al., 2007, IJSEM. 7:81-91

4. JSpecies (www.imedea.uib.es/jspecies) JSpecies  Biologist oriented  user friendly and usable with multifasta data

5. ANI is way to circumscribe species genomically in the future ANIm vs DDH:  85 genospecies evaluated  94-96% a plausible borderline  inconsistent results most probably due to wrong DDH values  ANI thresholds of 94-96%  genomospecies  20% random sequences (i.e., 250 nuc) of two genomes is enough  Complete catalogue of type strain genomes  only 4% random genome sequence is enough Richter & Rosselló-Móra 2009, PNAS 106: 19126-19131

6. The best scenario ◄► all species genomes sequenced afedcb glkjih mrqpon sxwvut complete type strain genomes + < 20% random sequence genome coverage Perhaps with 1000 reads would be enough (200€) STABLE ANI  1% of the genome will be enough for IDENTIFICATION purposes  need of an effort to full sequence the species collection  need of an effort to full sequence the species collection (GEBA; Wu et al. 2009 Nature 24: 1056-1060)  it will be in the future necessary to fully sequence any new type strain  94% - 96% ANI boundary

7. ► Data analysis in summer 2009 => 938 genomes ► 10% of the entries tagged with the collection number (the rest with original strain number) ► 255 species names represented by their Type Strain ► 256 species names NOT represented by their Type Strain ► 50 species names NEVER validly published ► it is possible to circumscribe uncultured species (i.e. Buchnera & Wolbachia) Richter & Rosselló-Móra 2009, PNAS 106: 19126-19131 Genome database & Type strains

8. Tetranucleotide variation: 4 4 = 256 TETRA:  Genomes have an oligonucleotide usage (not yet understood, related to codon usage)  Similar genomes might have similar usage  ALIGNMENT FREE PARAMETER  may be useful in deciding whether a group of strains deserve a species status  Same species >0.999

9. ► The case of the synthetic genome of M. mycoides strain GM12 transplanted to M. capricolum (Science (2010) 329: 52) ► 88.5 (66% aligned) ► 94.5 (78% aligned) ► 87.8 (76% aligned)

10. ► Only one of the several transplantations worked out! ► Different ways of reading the genome? organismtargetANI TETRA (r) M. hyopneumoniae 7448 M. hyopneumoniae J 98.20.999 M. mycoides LC M. capricolum 87.80.977 M. genitalium M. capricolum 63.40.620 M. genitalium M. pneumoniae 68.90.738 M. genitalium M. gallisepticum 64.10.622 M. aligatoris (crocodyli) M. capricolum 65.20.852 0.998 Same species 0.977 0.738 0.6220.852 WorkedNONONO Genome transplantation experiments of Venter

11. ► The phylogenetic (evolutive) distance plays an important role in the recognition of how the genetic information is coded ► M. genitalium  M. pneumoniae, strange! Wrong identified strain?

12. OTHER PARAMETERS Average Aminoacid Identity (AAI) Kostantinidis & Tiejde, 2005, J. Bacteriol. 187:6258-6264 Maximal Unique and Exact Matches (MUM) De Loger et al., 2009, J. Bacteriol. 191: 91-99 High Scoring Segment Pairs (HSP) (HSP) Auch et al., 2010. Std Gen Sci 2:117-134 And more to come Need full genome sequences The easiest is the best