Analyses of ORFans in microbial and viral genomes Journal club presentation on Mar. 14 Albert Yu

ORFan Defenition: an ORF with no detectable sequence similarity to other ORFs in the database considered Nearly all genomes have ORFans (df %) The more genomes sequenced, the more ORFans have found Most are annotated as hypothetical proteins of unknown function (no exp.)

ORFan continue More data… real, functional proteins 3D nstructure conserved in closely related species (Ka/Ks) Origin of ORFans ???????? Viral genome Microbial genome ? Viral laterally transferred genes (especially phages)

Viral genome Microbial genome

Question: the origin of ORFans Test hypothesis: ORFans have been acquired through lateral gene transfer from viruses To find homologs to these microbial ORFans within the virus sequence database

Genome-wide quantitative study BLASTP 277 microbial genomes 1456 viral genomes H(g): the number of genomes having at least one homolog of ORFan g U(g): uniqueness: the genomic distance between the genomes with ORFan g

Classification of ORFans Singleton: without any homolog wherever H=1, BLASTP=1 Paralogous: homologs in the same genome H=1, BLASTP>1 Orthologous: homologs within very closely related microbial genome H>1, U <= 0.1(by observations)

The U-value for all ORFs in prokaryote genomes In total: ORFs: ORFans: S: 64324(7.8%) P: 10419(1.3%) O: 35443(4.3%) 0.64 S or p O

ORFans-VH%(OVH): % of ORFans having homologs in viruses (0% ~ 63.8%) Non-ORFans-VH%(NOVH): % of non- ORFans having homologs in viruses (4.1% ~ 18.2%) The strength of the hypothesis = the value between these two VH%

Percentages of microbial ORFs with homologs in viruses Red: OVH Blue: NOVH 24 phylogenetic clades Bacteria Archea Firmicutes Gamma proteobacteria

The average % of OVH and NOVH in various groups % vs 9 % 8.5% vs 2.7 % 6.6% vs 0.8 %

Conclusion Most OVH << NOVH: current evidence supporting the hypothesis is weak Firmicutes and Gamma-proteobacteria have the highest number of homologs in viruses (viral database is biased) Viral database bias 1456 viruses 280 phages (109--Gamma; 102--Firmicutes; 69--others) Sampling ?????

Viral genome Microbial genome

277 Microbial genomes 1456 viruses All-virus-DB: ORFs 280 phages (20%) Phage-DB: ORFs (42%) ORFans: all-virus: 13078(30%) (v.s. all-virus-DB) 8200 (v.s. all nr, env-nr) all-phage: 6765 (v.s. all-virus-DB) 7047 (v.s. phage-DB)

Some characteristics of ORFans Bacterial ORFans are shorter than non- ORFans on average Bacterial ORFans have significant lower GC3 content than non-ORFans

The length of Viral ORFans and non-ORFans Length: Non-ORFans > ORFans

Length: ORFans < non-ORFans GC3%: ORFans < non-ORFans

The number of ORFs per genome in 1456 viruses Focusing on phage: higher %

The growing of the number of phage ORFans (consistent) Drop to 0 ? Keep increasing 38.4%

Each microbial species is a host for at least 10 phage species --- the phage diversity is at least 10 times higher than microbial diversity Only 280 phage genomes in database (low phage sampling)

Less than 5 phages Virus sampling bias between and within groups

The H-value percentages for all phage ORFs and prokaryotic ORFs prokaryotes phages 9.1% - ORFans 11.3% - ortho 38.4% - ORFans 32.4% - ortho

the H-value percentages of phage ORFs

4397(61.5%) / 7150(63.8%) / (prophage/ prokaryotic homologs/ phage non-ORFans) 589(44.7%) / 1317(18.7%) / 7047 (prophage/ prokaryotic homologs/ phage ORFans) 4987(58.9%)/8467(46.4%)/18248 (prophage/ prokaryotic homologs/ phage ORFs)