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The document discusses sequence similarity searching and comparison. It describes how programs like BLAST and FASTA are used to rapidly identify similarities between sequences and determine evolutionary relationships. BLAST and FASTA utilize word matching and heuristics to efficiently search large databases and return local or global alignments with scoring of matches. They provide a powerful method for functions prediction by comparing new sequences to known genes and proteins.

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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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