BIOINFORMÁTICA UFMG

A TGC

BIOINFORMÁTICA UFMG

A TGC Genômica e Bioinformática

ESTs mesmo que redundantesGenoma completo ou morte!

19952000

BIOINFORMÁTICA UFMG

A TGC O fim de uma EST

(A)200

AUG

(A)200(T)18cDNA (fita -)

AUG(A)18

cDNA (fita +)

cDNA (fita +)

(T)18cDNA (fita -)

(A)18

ATGATCATGACTTACGGGCGCGCGATxxxxxx

GGCGCGCGATATCCxxxx

AAATTTATTATCCxxxxx

3’EST

3’EST5’EST

5’EST

AAATTTATTATCCATCTACGxxxx

Uma foto de um novo transcriptoma [otorrin...] [...damonh...]

start

end

BIOINFORMÁTICA UFMG

A TGC Vida depois de PHRED 15

Query: 469 TTAGGAGGATCGTTTTTAGAATCCCCTGCAACGTTACCACGGTGGATTTCACTGACTGCG 528 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 1038 ttaggaggatcgtttttagaatcccctgcaacgttaccacggtggatttcactgactgcg 979

Query: 529 ACGTTCTTAACGTTGAATCCAACGTTGCTACCAgggagagcctcagtaagtgcttcatga 588 ||||||||||||||||| || |||||||||||||||||| ||||||||||||||||||||Sbjct: 978 acgttcttaacgttgaagcccacgttgctaccagggagaccctcagtaagtgcttcatga 919

Query: 589 tgcatttcgacagaattgacttcagtcgacaaaccttgcggagcaaaagtgacgaccata 648 |||||||||||||| |||||||||| |||| ||||||||||| |||||||||||||||||Sbjct: 918 tgcatttcgacagacttgacttcagccgaccaaccttgcggaccaaaagtgacgaccata 859

Query: 649 ccaggcttgatgataccagtttcaacgc 676 ||||||||||||||||||||||||||||Sbjct: 858 ccaggcttgatgataccagtttcaacgc 831

Query: non trimmed read. Subj: published sequence

BIOINFORMÁTICA UFMG

A TGC When PHRED meets BLAST

pUC18 (published sequence)

Sequencing reaction: single pool distributed over 3 96-well plates3 MegaBACE3 reads each - 846 reads total

Processing:MegaBLAST (BLASTn, SWAT)Phred– trim: a chromatogram analyzer– trim_alt: increasing trim_cutoff from 1% up to 25%

BIOINFORMÁTICA UFMG

A TGC O fim de uma EST

-500

-400

-300

-200

-100

0

100

200

1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% 16% 17% 18% 19% 20% 21% 22% 23% 24% 25%

Trim_cutoff parameter value(%)

Num

ber o

f bas

es

Included (trim) Discarded (trim) Included (TrimAlt) Discarded(TrimAlt)

PHRED 10 (10% error): only losses

BIOINFORMÁTICA UFMG

A TGC

BIOINFORMÁTICA UFMG

A TGC

0,00%

5,00%

10,00%

15,00%

20,00%

25,00%

30,00%

1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% 16% 17% 18% 19% 20% 21% 22% 23% 24% 25%

total miscall stepwise miscall

16% 17%

Trimmed reads

% errorin sequence

Added bases

3%

% errorin the tip

Error occurrence:

BIOINFORMÁTICA UFMG

A TGC Virtual pUC18 protein: STOP = *

>protein_puc18RQGFPSHDVVKRRPVPSLHACRSTLEDPRVPSSNS*SWS*LFPV*NCYPLTIPHNIRAGSIKCKAWGA**VS*LTLIALRSLPAFQSGNLSCQLH**IGQRAGRGGLRIGRSSASSLTDSLRSVVRLRRAVSAHSKAVIRLSTESGDNAGKNM*AKGQQKARNRKKAALLAFFHRLRPPDEHHKNRRSSQRWRNPTGL*RYQAFPPGSSLVRSPVPTLPLTGYLSAFLPSGSVALSHSSRCRYLSSV*VVRSKLGCVHEPPVQPDRCALSGNYRLESNPVRHDLSPLAAATGNRISRARYVGGATEFLKWWPNYGYTRRTVFGICALLKPVTFGKRVGSS*SGKQTTAGSGGFFVCKQQITRRKKGSQEDPLIFSTGSDAQWNENSR*GILVMRLSKRIFT*ILLN*K*SFKSI*SIYE*TWSDSYQCLISEAPISAICLFRSSIVA*LPVV*ITTIREGLPSGPSAAMIPRDPRSPAPDLSAINQPAGRAERRSGPATLSASIQSINCCREARVSSSPVNSLRNVVAIATGIVVSRSSFGMASFSSGSQRSRRVT*SPMLCKKAVSSFGPPIVVRSKLAAVLSLMVMAALHNSLTVMPSVRCFSVTGEYSTKSF*E*CMRRPSCSCPASIRDNTAPHSRTLKVLIIGKRSSGRKLSRILPLLRSSSM*PTRAPN*SSASFTFTSVSG*AKTGRQNAAKKGIRATRKC*ILILFLFQYY*SIYQGYCLMSGYIFECI*KNKQIGVPRTFPRKVPPDV*ETIIIMTLTYKNRRITRPFRLARFGDDGENL*HMQLPETVTACL*ADAGSRQARQGASAGVGGCRGWLNYAASEQIVLRVHHMRCEIPHRCVRRKYRIRRHSPFRLRNCWEGRSVRASSLLRQLAKGGCAARRLSWV

BIOINFORMÁTICA UFMG

A TGC tBLASTn (BLASTx) maximize with PHRED 8

Variação de score usando tblastn em sequências pUC 18 trimadas com phred em diversos valores de cutoff

% erro0 4 8 12 16 20 24

scor

e

0

100

200

300

400

500

15

8

Trim_cutoff parameter value (%)

BL

AST

x sc

ore

BIOINFORMÁTICA UFMG

A TGC Summarizing

PHRED meets BLAST as errors in tip are 16%Molecules carry 3% global errorAnd scores for EST vs aa comparisons maximizeReal life: crossmatch ends with X’sAuthors:– Fabiano Peixoto (CENAPAD)– Francisco Prosdocimi (Lab Biodiversidade)– Maurício Mudado (Lab Biodados)

BIOINFORMÁTICA UFMG

A TGC pUC18 proteina virtual