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Sequence Similarity Searching
Why Compare Sequences? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Are there other sequences like this one? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Similarity ≠ Homology ,[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],GATG C C A T A G A G C T G T A G T C GT A CCC T < — — > C T A G A G A G C - G T A G T C AG A GTG T CTTTGAGTTCC How to Compare Sequences?
Global vs Local similarity ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Search with Protein, not DNA Sequences ,[object Object],[object Object],[object Object],[object Object]
Similarity is Based on Dot Plots ,[object Object],[object Object],[object Object],[object Object]
Simple Dot Plot
Dot plot filtered with 4 base window and 75% identity
Dot plot of real data
Global vs. Local Alignments
Scoring Similarity ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
The PAM 250 scoring matrix
What program to use for searching? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA Algorithm
Makes Longest Diagonal ,[object Object],[object Object]
FASTA Alignments
FASTA Results - Histogram ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA Results - List ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA Results - Alignment ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA on the Web ,[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA Format ,[object Object],[object Object],[object Object],>URO1 uro1.seq Length: 2018 November 9, 2000 11:50 Type: N Check: 3854 .. CGCAGAAAGAGGAGGCGCTTGCCTTCAGCTTGTGGGAAATCCCGAAGATGGCCAAAGACA ACTCAACTGTTCGTTGCTTCCAGGGCCTGCTGATTTTTGGAAATGTGATTATTGGTTGTT GCGGCATTGCCCTGACTGCGGAGTGCATCTTCTTTGTATCTGACCAACACAGCCTCTACC CACTGCTTGAAGCCACCGACAACGATGACATCTATGGGGCTGCCTGGATCGGCATATTTG TGGGCATCTGCCTCTTCTGCCTGTCTGTTCTAGGCATTGTAGGCATCATGAAGTCCAGCA GGAAAATTCTTCTGGCGTATTTCATTCTGATGTTTATAGTATATGCCTTTGAAGTGGCAT CTTGTATCACAGCAGCAACACAACAAGACTTTTTCACACCCAACCTCTTCCTGAAGCAGA TGCTAGAGAGGTACCAAAACAACAGCCCTCCAAACAATGATGACCAGTGGAAAAACAATG GAGTCACCAAAACCTGGGACAGGCTCATGCTCCAGGACAATTGCTGTGGCGTAAATGGTC CATCAGACTGGCAAAAATACACATCTGCCTTCCGGACTGAGAATAATGATGCTGACTATC CCTGGCCTCGTCAATGCTGTGTTATGAACAATCTTAAAGAACCTCTCAACCTGGAGGCTT
BLAST Searches GenBank ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
BLAST ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
BLAST Algorithm
BLAST Word Matching ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Break query into words: Break database sequences into words:
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Compare Word Lists ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],? Compare word lists by Hashing (allow near matches)
ELEPRRPRYRVPDVLVADPPIARLSVSGRDENSVELT MEA T TDVRWMSETGIIDVFLLLGPSISDVFRQYASLTGTQALPPLFSLGYHQSRWNY IWLDI EEI HADGKRYFTWDPSRFPQPRTMLERLASKRRV KLV AIVDPH ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Find locations of matching words in database sequences
Extend hits one base at a time
HVTGRSAF_FS YYG YGCYC GLG TGKGLPVDATDRCCWA QSVFDYI YYG CYCGW GLG _GK__PRDA ,[object Object],[object Object],Query: Seq_XYZ: E-val = 10 -13
HSPs are Aligned Regions ,[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
 
The PAM 250 scoring matrix
BLAST has Automatic Translation ,[object Object],[object Object],[object Object]
BLAST Results - Summary
BLAST Results - List
BLAST Results - Alignment > gi|17556182|ref|NP_497582.1| Predicted CDS, phosphatidylinositol transfer protein [Caenorhabditis elegans] gi|14574401|gb|AAK68521.1|AC024814_1 Hypothetical protein Y54F10AR.1 [Caenorhabditis elegans] Length = 336 Score = 283 bits (723), Expect = 8e-75 Identities = 144/270 (53%), Positives = 186/270 (68%), Gaps = 13/270 (4%) Query: 48 KEYRVILPVSVDEYQVGQLYSVAEASKNXXXXXXXXXXXXXXPYEK----DGE--KGQYT 101 K+ RV+LP+SV+EYQVGQL+SVAEASK P++ +G+ KGQYT Sbjct: 70 KKSRVVLPMSVEEYQVGQLWSVAEASKAETGGGEGVEVLKNEPFDNVPLLNGQFTKGQYT 129 Query: 102 HKIYHLQSKVPTFVRMLAPEGALNIHEKAWNAYPYCRTVITN-EYMKEDFLIKIETWHKP 160 HKIYHLQSKVP +R +AP+G+L IHE+AWNAYPYC+TV+TN +YMKE+F +KIET H P Sbjct: 130 HKIYHLQSKVPAILRKIAPKGSLAIHEEAWNAYPYCKTVVTNPDYMKENFYVKIETIHLP 189 Query: 161 DLGTQENVHKLEPEAWKHVEAVYIDIADRSQVL-SKDYKAEEDPAKFKSIKTGRGPLGPN 219 D GT EN H L+ + E V I+IA+ + L S D + P+KF+S KTGRGPL N Sbjct: 190 DNGTTENAHGLKGDELAKREVVNINIANDHEYLNSGDLHPDSTPSKFQSTKTGRGPLSGN 249 Query: 220 WKQELVNQKDCPYMCAYKLVTVKFKWWGLQNKVENFIHKQERRLFTNFHRQLFCWLDKWV 279 WK + P MCAYKLVTV FKW+G Q VEN+ H Q RLF+ FHR++FCW+DKW Sbjct: 250 WKDSVQ-----PVMCAYKLVTVYFKWFGFQKIVENYAHTQYPRLFSKFHREVFCWIDKWH 304 Query: 280 DLTMDDIRRMEEETKRQLDEMRQKDPVKGM 309 LTM DIR +E + +++L+E R+ V+GM Sbjct: 305 GLTMVDIREIEAKAQKELEEQRKSGQVRGM 334
BLAST alignments are short segments ,[object Object],[object Object],[object Object]
BLAST 2 algorithm ,[object Object],[object Object],[object Object]
Web BLAST runs on a big computer at NCBI ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
FASTA/BLAST Statistics ,[object Object],[object Object],[object Object],[object Object]
BLAST is Approximate ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Interpretation of output ,[object Object],[object Object],[object Object],[object Object]
Biological Relevance ,[object Object],[object Object],[object Object],[object Object]
Borderline similarity ,[object Object],[object Object],[object Object],[object Object],[object Object]
Advanced Similarity Techniques ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
ESTs have frameshifts ,[object Object],[object Object],[object Object],[object Object],[object Object]
Genome Alignment ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Sim4 is for mRNA -> DNA Alignment ,[object Object],[object Object],[object Object],[object Object]
More Genome Alignment ,[object Object],[object Object],[object Object],[object Object]
Smith-Waterman searches ,[object Object],[object Object],[object Object],[object Object]
Smith-Waterman on the Web ,[object Object],[object Object],[object Object],[object Object]
Strategies for similarity searching ,[object Object],[object Object],[object Object]

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Similarity

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  • 10. Dot plot filtered with 4 base window and 75% identity
  • 11. Dot plot of real data
  • 12. Global vs. Local Alignments
  • 13.
  • 14. The PAM 250 scoring matrix
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  • 32. Extend hits one base at a time
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  • 37. The PAM 250 scoring matrix
  • 38.
  • 39. BLAST Results - Summary
  • 41. BLAST Results - Alignment > gi|17556182|ref|NP_497582.1| Predicted CDS, phosphatidylinositol transfer protein [Caenorhabditis elegans] gi|14574401|gb|AAK68521.1|AC024814_1 Hypothetical protein Y54F10AR.1 [Caenorhabditis elegans] Length = 336 Score = 283 bits (723), Expect = 8e-75 Identities = 144/270 (53%), Positives = 186/270 (68%), Gaps = 13/270 (4%) Query: 48 KEYRVILPVSVDEYQVGQLYSVAEASKNXXXXXXXXXXXXXXPYEK----DGE--KGQYT 101 K+ RV+LP+SV+EYQVGQL+SVAEASK P++ +G+ KGQYT Sbjct: 70 KKSRVVLPMSVEEYQVGQLWSVAEASKAETGGGEGVEVLKNEPFDNVPLLNGQFTKGQYT 129 Query: 102 HKIYHLQSKVPTFVRMLAPEGALNIHEKAWNAYPYCRTVITN-EYMKEDFLIKIETWHKP 160 HKIYHLQSKVP +R +AP+G+L IHE+AWNAYPYC+TV+TN +YMKE+F +KIET H P Sbjct: 130 HKIYHLQSKVPAILRKIAPKGSLAIHEEAWNAYPYCKTVVTNPDYMKENFYVKIETIHLP 189 Query: 161 DLGTQENVHKLEPEAWKHVEAVYIDIADRSQVL-SKDYKAEEDPAKFKSIKTGRGPLGPN 219 D GT EN H L+ + E V I+IA+ + L S D + P+KF+S KTGRGPL N Sbjct: 190 DNGTTENAHGLKGDELAKREVVNINIANDHEYLNSGDLHPDSTPSKFQSTKTGRGPLSGN 249 Query: 220 WKQELVNQKDCPYMCAYKLVTVKFKWWGLQNKVENFIHKQERRLFTNFHRQLFCWLDKWV 279 WK + P MCAYKLVTV FKW+G Q VEN+ H Q RLF+ FHR++FCW+DKW Sbjct: 250 WKDSVQ-----PVMCAYKLVTVYFKWFGFQKIVENYAHTQYPRLFSKFHREVFCWIDKWH 304 Query: 280 DLTMDDIRRMEEETKRQLDEMRQKDPVKGM 309 LTM DIR +E + +++L+E R+ V+GM Sbjct: 305 GLTMVDIREIEAKAQKELEEQRKSGQVRGM 334
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