Alignments in Practice BLAST and CLUSTAL Introduction to - - PowerPoint PPT Presentation

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Alignments in Practice BLAST and CLUSTAL Introduction to - - PowerPoint PPT Presentation

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Alignments in Practice BLAST and CLUSTAL Introduction to Bioinformatics Dortmund, 16.-20.07.2007 Lectures: Sven Rahmann Exercises: Udo Feldkamp, Michael Wurst 1 Overview Dot Plots Nucleotide BLAST Protein BLAST BLAST

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Alignments in Practice BLAST and CLUSTAL

Introduction to Bioinformatics Dortmund, 16.-20.07.2007 Lectures: Sven Rahmann Exercises: Udo Feldkamp, Michael Wurst

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Overview

  • Dot Plots
  • Nucleotide BLAST
  • Protein BLAST
  • BLAST Statistics
  • BLAT
  • CLUSTAL
  • JalView
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Dotter – Tool for Dot Plots

  • http://www.cgb.ki.se/cgb/groups/sonnhammer/Dotter.html
  • Dotlet: a Java applet for Dot Plots
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Dot Plots

  • Hemoglobin Alpha against Hemoglobin Beta
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EBI Alignment Service

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BLAST

  • URL: http://www.ncbi.nlm.nih.gov/BLAST/
  • Basic Local Alignment Search Tool
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Choose the right BLAST

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Nucleotide BLAST Interface

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BLAST Parameters

  • Expect threshold:

low [0.01] = strict high [100] = loose

  • Word size:

speed vs. sensitivity high = faster low = slower, but more sensitive

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Protein BLAST

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Protein BLAST Parameters

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Translated BLAST

  • protein query against nucleotide database

– nucleotide sequence not unique – also consider reverse complement

  • nucleotide query against protein database

– consider all 6 reading frames

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BLAST Output

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BLAST Output II

Database + Accession Link Bit score E-value Description

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  • How good / reliable is a hit found by BLAST?
  • Raw score :=

score of the alignment according to scoring matrix and gap penalties

  • Bit score :=

score (log2 units), length-normalized

  • E-value :=

Number of hits of such or better score in a hypothetical database of random proteins of the same size

BLAST Statistics

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More on Statistics

  • Null model :=

random model describing sequences without intentional signal (here: pair of random sequences without intentional similarity)

  • (single) p-value for observed score s :=

Prob(Score >= s) in the null model

  • (multiple) p-value :=

Prob(Score >= s at least once)

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BLAT

  • BLAST-Like Alignment Tool
  • index-based
  • developed at UC Santa Cruz
  • especially for searching in whole genomes
  • very fast
  • limited to nearly exact matches
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UCSC Genome Browser + BLAT

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CLUSTAL

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What Clustal Did (“Output file”)

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Clustal Results (pretty)

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Clustal Results (“alignment file”)

CLUSTAL W (1.83) multiple sequence alignment FOS_RAT MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNTQDFCADLSVSSANF 60 FOS_MOUSE MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNTQDFCADLSVSSANF 60 FOS_HUMAN MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNAQDFCTDLAVSSANF 60 FOS_CHICK MMYQGFAGEYEAPSSRCSSASPAGDSLTYYPSPADSFSSMGSPVNSQDFCTDLAVSSANF 60 FOS_ZEBRAFISH MMFTSLNADCDASS-RCSTASPSGDSVGYY------------PLNQTQEFTDLSVSSASF 47 **: .: .: :*.* ***:***:***: ** *:* : :**:****.* FOS_RAT IPTVTAISTSPDLQWLVQPTLVSSVAPSQTRAPHPYGLPTPS-TGAYARAGVVKTMSGGR 119 FOS_MOUSE IPTVTAISTSPDLQWLVQPTLVSSVAPSQTRAPHPYGLPTQS-AGAYARAGMVKTVSGGR 119 FOS_HUMAN IPTVTAISTSPDLQWLVQPALVSSVAPSQTRAPHPFGVPAPS-AGAYSRAGVVKTMTGGR 119 FOS_CHICK VPTVTAISTSPDLQWLVQPTLISSVAPSQNRG-HPYGVPAPAPPAAYSRPAVLKAP-GGR 118 FOS_ZEBRAFISH VPTVTAISSCPDLQWMVQP-MISSAAPS-------NGAAQSYNPSSYPKMRVTGAK---- 95 :*******:.*****:*** ::**.*** * . ..:*.: : : FOS_RAT AQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSAL 179 FOS_MOUSE AQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSAL 179 FOS_HUMAN AQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSAL 179 FOS_CHICK GQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEEEKSAL 178 FOS_ZEBRAFISH --TSNKRSRSEQLSPEEEEKKRVRRERSKMAAAKCRNRRRELTDTLQAETDQLEDEKSAL 153 : .:*.: **********:*:****.**************************:***** FOS_RAT QTEIANLLKEKEKLEFILAAHRPACKIPNDLGFPEE----MSVTS-LDLTGGLPEATTPE 234 FOS_MOUSE QTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPEE----MSVAS-LDLTGGLPEASTPE 234 FOS_HUMAN QTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPEE----MSVAS-LDLTGGLPEVATPE 234 FOS_CHICK QAEIANLLKEKEKLEFILAAHRPACKMPEELRFSEE----LAAATALDLG----APSPAA 230 FOS_ZEBRAFISH QNDIANLLKEKERLEFILAAHKPICKIPADASFPEPSSSPMSSISVPEIVTTSVVSSTPN 213 * :*********:********:* **:* : *.* :: : :: :..

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Clustal Guide Tree

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Clustal Guide Tree

  • Guide Tree is not a phylogenetic tree,

just a computational device

  • Cladogram: edge lengths have no meaning
  • Phylogram: edgle lengths correspond to

distances

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JalView: Alignment Editor (start from the CLUSTAL web site)

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Simple JalView Window

  • Simple alignment editor (Java applet)
  • Complex alignment editor (Java application)

– Web Start, or – Download installer

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Starting or Installing JalView

www.jalview.org

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Multiple Alignment @ BiBiServ

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For Windows/MAC: QAlign2

  • URL: http://gi.cebitec.uni-bielefeld.de/QAlign/
  • Live Demo of QAlign2