genome filtering with iBLASTgenomesAquifexaeolicusMethanococcusjannaschiiE.coligenomePyrococcus furiosisHalobacteriumsalinarumNRC-1Sulfolobussolfataricus医DeinococcusradioduransSynechocystissp.PCC6803BacillussubtilisEscherichiacoli通广AgrobacteriumtumefaciensUWashisolate100mersPseudomonasaeruginosa50bpoverlapSaccharomycescerevisiaeDeinococcusradioduransCaenorhabditiselegansblastnvgenomexparse,calcstats,sortbuild contigsblastnvntfilteredsetof100mersconservedsequences
genome filtering with iBLAST E. coli genome 100mers 50 bp overlap filtered set of 100mers blastn v genome x parse, calc stats, sort build contigs blastn v nt conserved sequences Aquifex aeolicus Methanococcus jannaschii Pyrococcus furiosis Halobacterium salinarum NRC-1 Sulfolobus solfataricus Deinococcus radiodurans Synechocystis sp. PCC 6803 Bacillus subtilis Escherichia coli Agrobacterium tumefaciens UWash isolate Pseudomonas aeruginosa Saccharomyces cerevisiae Deinococcus radiodurans Caenorhabditis elegans genomes
results at (bits>29)126sequencescomprisingrRNA (77)4regionsof16S;7regionsof23S(x716S/23Sloci)tRNA (53)non RNAABCtransporterATPbindingdomains(7)glnQ (2),ycbE,ddpF,yecC,hisP,yhdZothernuoM, upp+3'UTR, ygfG, yhaO+5'UTR, degQ, argR, ydhZ, selB/selA, yihl+3'UTRmicrobialonly(withoutusingS.cerevisiae&C.elegans)adds116Sregionatbp230-342(x716Sloci),2tRNA,&2ABCtransporterATPbindingdomains
results at (bits > 29) 126 sequences comprising: rRNA (77) 4 regions of 16S; 7 regions of 23S (x7 16S/23S loci) tRNA (53) non RNA ABC transporter ATP binding domains (7) glnQ (2), ycbE, ddpF, yecC, hisP, yhdZ other nuoM, upp+3’ UTR, ygfG, yhaO+5’ UTR, degQ, argR, ydhZ, selB/selA, yihI+3’ UTR microbial only (without using S. cerevisiae & C. elegans) adds 1 16S region at bp 230-342(x7 16S loci), 2 tRNA, & 2 ABC transporter ATP binding domains
tier 1: seguences resulting at > 38 bitsribosomalRNAs(bits)score16S23Scolor5960conservation555553325325001000150016S rRNA (nt)500100015002000250023S rRNA (nt)
0 500 1000 1500 2000 2500 23S rRNA (nt) 0 500 1000 1500 16S rRNA (nt) score (bits) 23S 16S color 60 59 55 55 52 52 39 39 31 31 29 29 tier 1: sequences resulting at > 38 bits ribosomal RNAs c o n s erv atio n
Usingshort16SgeneprimersandDM2polymerasetodetect divergent 16Sgenes anddivergent organisms1.computation identified ~10 nt'core'conserved regionsof 16S gene·fall within canonical universal primerbinding sites?shorterprimers increase theavailabletargets for16SPCR~1000x2.DM2isaPCRenzymeengineeredbyMJRtoprimefromshortprimersWillextend10merswitha SSB regionadded to TagDNApolymerase
Using short 16S gene primers and DM2 polymerase to detect divergent 16S genes and divergent organisms 1. computation identified ~10 nt ‘core’ conserved regions of 16S gene •fall within canonical universal primer binding sites •shorter primers increase the available targets for 16S PCR ~1000x 2. DM2 is a PCR enzyme engineered by MJR to prime from short primers, with a SSB region added to Taq DNA polymerase. Will extend 10 mers
DM2polymerase8F/338R338RCTGCTGCCTCCCGTAGGAGT2338R-18CTGCTGCCTCCCGTAGGA3338R-16CTGCTGCCTCCCGTAG4338R-14CTGCTGCCTCCCGT338R-12CTGCTGCCTCCCo338R-10CTGCTGCCTC515F/1391R515FGTGCCAGCMGCCGCGGTAA2515F-17GTGCCAGCMGCCGCGGT3515F-15GTGCCAGCMGCCGCG4515F-13GTGCCAGCMGCCG5515F-11GTGCCAGCMGC6515F-9GTGCCAGCM1515F-7GTGCCAG338Rlength(nt)515Flength(nt)20181614121019171513119515F/1391R8F/338R
DM2 polymerase 8F/338R 515F/1391R 338R length (nt) 20 18 16 14 12 10 515F length (nt) 19 17 15 13 11 9 7 8F/338R 1 338R CTGCTGCCTCCCGTAGGAGT 2 338R-18 CTGCTGCCTCCCGTAGGA 3 338R-16 CTGCTGCCTCCCGTAG 4 338R-14 CTGCTGCCTCCCGT 5 338R-12 CTGCTGCCTCCC 6 338R-10 CTGCTGCCTC 515F/1391R 1 515F GTGCCAGCMGCCGCGGTAA 2 515F-17 GTGCCAGCMGCCGCGGT 3 515F-15 GTGCCAGCMGCCGCG 4 515F-13 GTGCCAGCMGCCG 5 515F-11 GTGCCAGCMGC 6 515F-9 GTGCCAGCM 7 515F-7 GTGCCAG