VISTA family of computational tools for comparative genomics How can we leverage genome sequences from many species to learn about genome function?How can we leverage genome sequences from many species to learn about genome function? Microbial applicationsMicrobial applications Inna Dubchak, Genomics Division LBNL, JGI

Human Genome Annotation Gene A only 1–2% codingonly 1–2% coding efficient identification of regulatory sequences?efficient identification of regulatory sequences?

Sequence conservation implies function AGTTGAAAC GGAGCTGATGGAGC GGTGGGC T TACATTTCG ACTGTATCGCCTCG CAACCCT A potential functional region conservation sequence CTATAAATGC CTATAAATGC AC AC Last Common Ancestor divergence= non functional functional region =conservation 80 million years

Comparative Genomics Introduction Human Drosophila Mouse Urchin Chimp Similar Genes Synteny Sequence Alignment

VISTA is an integrated system for global sequence alignment and visualization for comparative genomic analysis

Algorithm Feature AVID *can handle draft sequence LAGAN ** produces true multiple alignments Shuffle-LAGAN ** handles rearrangements (inversions, translocations) * Lior Pachter, UC Berkeley ** Michael Brudno, U. Toronto How does VISTA Work: Global Genomic Aligments sequence 1 sequence 2 1- anchoring: identify regions of strong similarity 2- chaining: join regions of weak or no similarity

TCCCCAACTATAAATGGATGAAATTGCAGGAAATGACAGGTA-----TGACCCCTTCTCT >>>>>>>>> ||| ||| | |||||| | || || | | | ||||||| || <<<<<<<<< TCCTCAATTCAGAATGGAGGGAAGCACACAGGACACAGAGATCCCTTTACCCCCTTCGCT ACCAGAGGCTTGGATTTTTTTTCTTCTTCTCCTCCCTTAGCCCGTGTTGAGCTATTTCGG >>>>>>>>> | | | || | | | <<<<<<<<< ATGT TATCAGGCCACTCAAG AGTTTCCTGGCAGGGAAGAGCGAGTGAGGCTGCCTTACCTTCAGGATGACCACTAGCAGG >>>>>>>>> |||| | || || | ||||| ||||||| | ||| ||||||| ||||||||| |||||| <<<<<<<<< AGTTCCTTGTCAAG-AAGAGTGAGTGAGTCCACCTCACCTTCAAGATGACCACCAGCAGG CCAGCGCTCACAAGAAGAGGAATGAGGCTACTAATGAACCAGCTAAACCAGAGGATGCTG >>>>>>>>> |||||||||||||| ||||| |||||||| |||| |||||||||||||||||||||| <<<<<<<<< CCAGCGCTCACAAGCAGAGGGATGAGGCTGCTAACAAACCAGCTAAACCAGAGGATGCCA TTGTCCAGGCCCATGATCCGCATGGTCTCTTTCAGCCGTGCCTCCTTCTCATACACGATG >>>>>>>>> |||||||| |||||||||||||||||||| |||||||| ||||||||||||||||| ||| <<<<<<<<< TTGTCCAGACCCATGATCCGCATGGTCTCCTTCAGCCGAGCCTCCTTCTCATACACAATG CCCTTGATGATCACAGCCACTGAGTAAATCCAGGCCAGCGTCATGAAGAGGGGCATTGAC >>>>>>>>> | ||||||||||||||| || ||||| |||||||| || ||||||||||||||||||||| <<<<<<<<< CTCTTGATGATCACAGCGACAGAGTAGATCCAGGCTAGAGTCATGAAGAGGGGCATTGAC CGGCTCATCACCCGCAGAAAGCTGGAGGCCCCAAGGAAGGACAAGGGGAGAAAGAAAGAC >>>>>>>>> |||||||| ||||||||||| |||||||| | || || | || ||| | || |||| <<<<<<<<< CGGCTCATGACCCGCAGAAAACTGGAGGCACAGAGAAAAGGCATGGGAAAAATGAAAAGT ACACGTGAGCCAGGGTGATGGGCCAAGGCCTCTGAGCCTGCATGCTAGAGGGAGCACCAC >>>>>>>>> ||||||| || | ||||||||| |||| || |||| ||| | <<<<<<<<< GTGAGCCCGG-CACCGATCCAAGGCCT TGCACACTGGAGGACAAACCTC ATCTGGGCCACAGAAGGACAGGCCCTCTAGACTCTGAAATGTACGTATGATCCAATGCTT >>>>>>>>> ||| ||| | | | | | |||||| || ||||| ||||| | | || | || <<<<<<<<< ATCAGGGTCGCTTATGAA-AGGCCCACTGAACTCTCAAATG ACCAAAGGTTT CACGAGCAATGCAATGTAGAGAGAAAAACGAGGCTAACAAAGTGTTGCCAAACCAAATTT >>>>>>>>> || |||| || | ||||| ||| | || | | || | ||| | |||||| <<<<<<<<< CATTAGCAGTGGA---CAGAGATGAAACCTGGGTTTCGAGGGTATGGCCGTGCAAAATTT CTTTGGGGGCTTGCTTCAGTAACTAGGTAACTGTGAGCGATAC-TTAAACTAAAGGTAGA >>>>>>>>> || |||||| ||| | || ||||| || | || | | |||| |||| || <<<<<<<<< TTTCAGGGGCTCTCTTTAATAGCTAGGAAATGGATAGGGTAATATTAAGATAAATATAAG TTATGTTA--AAGTACTAAAAACCAAAACA------AAAAAACAACTCATTCTCTCACAA >>>>>>>>> ||| || |||||||||| || || | || ||||| ||| | | | <<<<<<<<< TTACTCTACTAAGTACTAAACACAAAGGGCGGGGGCAGAATCCAACTTGGTCTTCCGCTA Global Genomic Aligner Output

VISTA visualization GTAGTGCCACTGAGTGTGACAGGGATGGCAAGAAAAGCATTAAGTTCCAAGGGGAAAGAA >>>>>>>>> | || ||| ||| |||| |||||||||| | || || |||| | |||||||| <<<<<<<<< GAGATGTCACCAAGTA-AACAGAGATGGCAAGAGGACCAATAGGTTCTAGTGGGAAAGAC “sliding window” to measure sequence conservation (default window size 100bp) Graphical presentation of sequence conservation as “peaks-and-valley” curve >70% identity base sequence coordinates % identity

VISTA homepage: VISTA Servers (submit your own data) VISTA Browsers (precomputed alignments) Other VISTA-related Projects Access servers, browsers, other information

wgVISTA Align and compare sequences, including microbial assemblies mVISTA Align and compare sequences rVISTA Search for TFBS combined with a comparative sequence analysis VISTA Servers GenomeVISTA Align DNA sequence to a genome

VISTA Browser Browse through pre-computed whole-genome alignments Whole Genome rVISTA Whole genome analysis for conserved TFBS over-represented in upstream regions of genes Precomputed Alignments VISTA-Point Browse and obtain sequence and alignment data

VISTA Browser: Access

VISTA Browser: Input Menu genomeposition visualization Java 2, if needed Choose “base” genome Select location Determine visualization preference VISTA Browser VISTA tracks on UCSC Browser VISTA-Point

VISTA Browser: Alignment Details direction exon repeats alignment SNPs gene

VISTA Browser: Result Position on chromosome Control Panel Graphical display of genome alignments Color Legend Cursor Info Menu & Icons Curve annotation (species) 1 row

VISTA Browser: Zooming vs. rhesus vs. dog

VISTA browser

VISTA Point: Access Overview

VISTA Point: Graphics Table

VISTA Point: AlignmentsTable sequence

Google map-like Dot-Plot

BlockView – Synteny Plot tool

RegTransBase – experimental data manually curated database of regulatory interactions captured from literature; 6000 papers RegPrecise – computational predictions manually curated database of regulons inferred by comparative genomics approach RegPredict – web tool for regulon inference integrated system for fast and accurate inference of regulons by comparative genomics NAR database issue, 2010; Featured Article NAR Web Server issue, 2010; Featured Article Principal components NAR database issue, 2007

mVISTA: Access

mVISTA: Interface Our example will show 3 sequences Align up to 100 sequences

mVISTA: Input of Sequences Provide your address Upload your sequences Or enter GenBank ID your upload file or GenBank ID

AVID multiple pair wise alignments accepts finished or draft sequences LAGAN true multiple alignments mVISTA: Input Parameters  Shuffle-LAGAN –multiple pair wise alignments –detects sequence rearrangements and inversions

mVISTA: Results PDF VISTA Browser VISTA -Point

wgVISTA: Microbial Assemblies Comparison wgVISTA: whole genome VISTA Compares 2 sequences (up to 10 Mb) Draft or finished microbial assembly sequences can be used

rVISTA: Access

Regulatory VISTA (rVISTA): prediction of transcription factor binding sites Simultaneous searches of the major transcription factor binding site database (Transfac) and the use of global sequence alignment to sieve through the data rVISTA search is automatically run when submitting: mVISTA mVISTA genomeVISTA genomeVISTA

Human TGATTTCTCGGCAGCAAGGGAGGGCCCCATGACAAAGCCATTTGAAATCCCAGAAGCAATTTTCTACTTACGACCTCACTTTCTGTTGCTGTCTCTCCCTTCCCCTCTG Mouse TGATTTCTCGGCAGCCAGGGAGGGCCCCATGACGAAGCCACTCGAAATCCCAGAAGCAATTTTCTACTTACGACCTCACTTTCTGTTGCTCTCTCTTCCTCCCCCTCCA Dog TGATTTCTCGGCAGCAAGGGAGGGCCCCATGACGAAGCCATTTGAAATCCCAGAAGCGATTTTCTACCTACGACCTCACTTTCTGTTGCGCTCACTCCCTTCCCCTGCA Rat TGATTTCTCGGCAGCCAGGGAGGGCCCCATGACGAAGCCACTCGAAATCCCAGAAGCAATTTTCTACTTACGACCTCACTTTCTGTTGTTCTCTCTTCCTCCCCCTCCA Cow TGATTTCTCGGCAGCCAGGGAGGGCCCCATGACGAAGCCATTTGAAATCCCAGAAGCAATTTTCTACTTACGACCTCACTTTCTGTTGCGTTCTCTCCCTTCCCCTCCT Rabbit TGATTTCTCGGCAGCCAGGGAGGGCCCCACGAC-AAGCCATTCAAAATCCCAGAAGTGATTTTCTACTTACGACCTCACTTTCTGTTG----CTCTCTCCTTCCCTCCA Ikaros-2 Ikaros-2 NFAT Ikaros-2 20 bp dynamic shifting window >80% ID 1. Identify potential transcription factor binding sites for each sequence using library of matrices (TRANSFAC) 2. Identify aligned sites using VISTA 3. Identify conserved sites using dynamic shifting window Regulatory VISTA (rVISTA):

rVISTA: Interface your sequences rVISTA sequence submission: set number Submit address, sequences, and set parameters Key step: click the box for: Find potential transcription factors

rVISTA: Select TRANSFAC Matrices

rVISTA: Mailed Results ed results will provide a link Choose which binding sites matrices to display You can then choose visualization options display

rVISTA: Results Graphic Blue all transcription factor (TF) binding sites Red TF sites which are aligned in both sequences Green TF sites which are aligned & in conserved regions sequences sites

Whole Genome rVISTA: Access

Whole Genome rVISTA: Select Alignment IDs or symbols upstream range

Whole Genome rVISTA: Results sites found view genes

Examples of VISTA usage Non-coding regulatory regions, for example enhancers Genes from the same gene families Alternative splicing Transcriptional regulation Genetic studies References collected are available through the Publications link at the VISTA home page

VISTA-related Publications

VISTA thanks BiologyGenomics Division, LBNL lead by Dr. Edward Rubin Dario BoffelliKelly Frazer Gaby Loots Len PennacchioMarcelo Nobrega Axel Visel Bioinformatics Michael BrudnoOlivier Couronne Simon Minovitsky Igor RatnerAlexander Poliakov Lior Pachter (UCB) Shyam PrabhakarDmitriy RyaboyNameeta Shah Inna Dubchak