Comparative Sequence Analysis in Molecular Biology. Martin Tompa Computer Science & Engineering Genome Sciences University of Washington Seattle, Washington, U.S.A. Outline. What genome data is available? What is phylogenetic footprinting?
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Comparative Sequence Analysis in Molecular Biology
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Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
How Many Genomes Are Available? • 46 vertebrate genomes sequenced (primates to rodents to marsupials to birds to fishes) • 1766 bacterial genomes sequenced (as of 2/12/2012) • Insects, fungi, worms, plants, … • Many more will be finished very soon • Fertile ground for comparative genomics
Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
Phylogenetic Footprinting(Tagle et al. 1988) • Functional regions of DNA (regions under “purifying constraint”) evolve slower than nonfunctional ones. • Consider a set of corresponding DNA sequences from related species. • Identify unusually well conserved subsequences (i.e., ones that have not mutated much over the course of evolution): “motifs”
Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
How to Find Conserved Motifs ACTAACCGGGAGATTTCAGAhuman AAGTTCCGGGAGATTTCCAchimp TAGTTATCCGGGAGATTAGAmouse AAAACCGGTAGATTTCAGGrat
Multiple Sequence Alignment AC--TAACCGGGAGATTTCAGA human AAGTT--CCGGGAGATTTCC-Achimp TAGTTATCCGGGAGATT--AGAmouse AA---AACCGGTAGATTTCAGGrat (Finding the optimal alignment is NP-complete.)
Phylogenetic Footprinting • Use whole-genome multiple alignment such as provided by UCSC Genome Browser. • Search for regions of well conserved alignment. • Regulatory elements [Cliften; Kellis; Kolbe; Prakash; Woolfe; Xie (2)] • RNA elements [Pedersen; Washietl] • General conservation & constraint [Bejerano; Boffelli; Cooper; Margulies (4); Pollard; Prabhakar; Siepel]
Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
Why Doubt Alignments? • Multiple sequence alignment of short sequences (proteins, promoters) is difficult (NP-complete) • Aligning whole genomes adds the complications of huge sequences and genomic rearrangements • Vertebrate alignment has 3.8 billion columns • Automatically generated
Assessing 4 Genome-Size Alignments(with Xiaoyu Chen) • Alignments: MLAGAN [Brudno 2003], MAVID [Bray 2003], TBA [Blanchette 2003], Pecan [Paten 2008] • Target ENCODE regions: 30 Mbp covering 1% of the human genome (ENCODE targets) • Total input: 554 Mbp over 28 vertebrates • Rich resource for comparing and assessing genome-size alignments Margulies et al. 2007, Genome Research
Alignment agreement for mouse • Intronic & intergenic account for 95% of mouse bases aligned to human • Agree% in those categories: 44% to 62% • Much worse for more distant species • Building reliable MSA remains challenging Intronic bases Intergenic bases Disagree% Unique% Agree%
Which Alignment Columns to Trust?(with Amol Prakash, generalizing Karlin and Altschul 1990) Goal: label each alignment column with confidence measure of alignment correctness • Identify sequences that do not belong • Users forewarned about regions of interest • Genome browser designers consider realigning • Alignment tool designers get feedback for possible improvements
Sample Suspicious Alignment Human -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Chimp -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Rhesus -----------GTTGCCATGC-AAAAATATTATGTCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Mouse -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CGTGTCAA----------TTAACAC Rat -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CGTGTCAA----------TTAACAC Dog -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Cow -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Elephant -----------GTTGCTATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Tenrec -----------GTTGCCATAC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATGTCAA----------TTAACAC Opossum -----------GTTGCCATGC-AAAAATATTATGGCTTTACTAAAATTTATACAAG---CATATCAA----------TTAACAC Chicken -----------GTTGCCATGCAAAAAATAATATGGCTTTACTAAAATTTACACAAC---CCTGACAA----------TTAACAC ZebrafishGAACATATCCGAGTGCTGTAA-AATACTACTGGGA----ACCAGAAATG—-ACAAGTTCCATGACAGCTTTGCCTTTTTGGCTC
Outline of Computation Input Multiple sequence alignment A For each branch k of the tree { Compute scoring function sck (Felsenstein) Find all maximally scoring segments of A usingsck(Ruzzo & Tompa) Compute K, using sck (Karlin & Altschul) Compute p-value pk of each segment score using K, (Karlin & Altschul) } Output Discordance: maxkpk
Suspicious Alignment Regions Back to four ENCODE alignments spanning 30 Mbp of human aligned to 27 other vertebrates (with Xiaoyu Chen) • Identify suspicious alignment regions: • Length 50 bp • Discordance 0.1 at each position, all with respect to the same worst species • Fewer than 50% gapped sites • Suspicious% • Percentage of aligned bases in suspicious regions
Can suspicious alignments be improved? • Baboon and MLAGAN (for example): all points (x,y), where • x = human-baboon alignment score of MLAGAN region suspicious for baboon • y = human-baboon alignment score of alternative alignment for same human region but not suspicious for baboon • y = x • y - x = μ, where μ = average y-xover all points • y - x = μ ± σ, where σ = standard deviation of y-xover all points
Conclusions • Disturbing lack of agreement among alignments: alignment still a hard problem • Performance of the aligners varies significantly by species group and region type, particularly distant species and noncoding regions
Outline • What genome data is available? • What is phylogenetic footprinting? • Phylogenetic footprinting by multiple sequence alignment • Which parts of multiple sequence alignments are trustworthy? • FootPrinter: phylogenetic footprinting without alignment
DNA TCCAACGGTGCTGAGGTGCAC Protein Gene DNA, Genes, and Proteins DNA: program for cell processes Proteins: execute cell processes
Regulation of Genes • What turns genes on and off? • When is a gene turned on or off? • Where (in which cells) is a gene turned on? • How many copies of the gene product are produced?
Regulation of Genes Transcription Factor RNA polymerase DNA Gene Regulatory Element
Regulation of Genes Transcription Factor RNA polymerase DNA Gene Regulatory Element
Goal • Identify regulatory elements in DNA sequences. These are: • Binding sites for proteins • Short subsequences (5-25 nucleotides) • Up to 1000 nucleotides (or farther) from gene • Inexactly repeating patterns (“motifs”)
