GENOMICA - Unifem.docente.unife.it/.../genomica-1/Prokopenko_Lezione1.pdfIl Progetto Genoma Umano...

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GENOMICA

Uso di Genome Browser per l'annotazione di sequenze genomiche.

UCSC

University of California Santa Cruz

Genome Browser

Dott.ssa Inga Prokopenko

Sequenziamento del genoma –assemblaggio delle sequenze disponibili

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� Una sequenza viene detta “finita” quando presenta un livello di errore inferiore a 1/10000 basi e non ha gaps.

� Il Progetto Genoma Umano era complesso dal punto di vista tecnico ma anche dal punto di vista computazionale.

� L’output di una singola reazione di sequenza (read) = 500-800 bp � Tutti i singoli frammenti dovevano essere assemblati in una singola stringa lineare.

Biological Databases

� L’annotazione del genoma è un’area di investigazione attiva ed include molte organizzazioni che pubblicano i risultati nella data banche biologiche disponibili per tutta la comunità:

� ENCyclopedia Of DNA Elements (ENCODE)� Entrez Gene� Ensembl� Gene Ontology Consortium� GeneRIF� RefSeq� Uniprot� Vertebrate and Genome Annotation Project (Vega)

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Browser genomici

� aiutano a visualizzare genomi annotati completi (inclusi geni e loro strutture, proteine, espressione, regulazione, variazione ed analisi comparative)

� Le risorse di annotazione sono multiple.� Genome Browsers� UCSC Genome Bioinformatics Genome Browser and

Tools (UCSC)� NCBI-UniGene (National Center for Biotechnology

Information) � Ensembl The Ensembl Genome Browser (Sanger

Institute and EBI)

Copyright OpenHelix. No use or reproduction without express written consent 4

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3 milioni di basi in formato testo = nessuna utilita’Servono:•Annotazione dell’informazione sulla sequenza•Possibilita’ di recuperare velocemente la sequenza di regioni specifiche del genoma in base a criteri di

• Contenuto di informazione• Caratteristiche di sequenza

Genomi disponibili

HumanHomo sapiens assembly• 99% delle regioni contenenti geni• accuratezza 99.99% • 2.84 Gb finite “highly contiguous”

SpeciesA. gambiaeA. melliferaC. briggsaeC. elegansC. intestinalisChickenChimpCowD. ananassaeD. erectaD. grimshawiD. melanogasterD. mojavensisD. persimilisD. pseudoobscuraD. sechelliaD. simulansD. virilis

UCSC Genome BrowserSistema per la “navigazione” della sequenza e dell’annotazione di genomi, che permette la visualizzazione dell’informazione a “diverso ingrandimento” ed il recupero di porzioni di sequenza con associate le informazioni di annotazione, come:Geni noti e geni predettiESTs, mRNAsIsole CpGassembly gaps e coverage, bande cromosomicheOmologia con altri genomi…

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Organizazione dei dati genomici…

Genome backbone: base position numbersequenzaA

nnot

atio

n T

rack

schromosome band

known genes

predicted genes

evolutionary conservation

SNPs

sts sites

gap locations

repeated regions

microarray/expression data

more…

Links out to more data


The UCSC Home page: genome.ucsc.edu

navigate

navigateGeneral information

Specific information—new features, current status, etc.


A sample of what we will find:

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The Genome Browser Gatewaystart page, basic search

text/ID searches

Helpful search examples,

suggestions below

� Use this Gateway to search by:� Gene names, symbols

� Chromosome number: chr7, or region: chr11:1038475-1075482

� Keywords: kinase, receptor

� IDs: NP, NM, OMIM, and more…

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UCSC Genome BrowserMolte possibilita’ per la ricerca di una regione spe cifica:

• chr7 un cromosoma intero• 20p13 una regione (banda p13 del cr. 20)• chr3:1-1000000 il primo milione di basi del cr. 3 dal ptel• D16S3046 regione intorno al marcatore (100,000 basi per lato)• RH18061;RH80175 regione tra i due marcatori• AA205474 regione genomica che si allinea con la sequenza con

questo GB accession number• PRNP regione del genoma che comprende il gene PRNP • NM_017414 regione del genoma con indificatore di RefSeq• NP_059110 regione del genoma con “protein accession number”• 11274 (LLID)

Oppure di liste di regioni:• pseudogene mRNA Lists transcribed pseudogenes, but not cDNAs• homeobox caudal Lists mRNAs for caudal homeobox genes• zinc finger Lists many zinc finger mRNAs• huntington Lists candidate genes associated with Huntington's

disease

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The Genome Browser Gatewaystart page choices, February 2005

Make your Gateway choices:1. Select Clade2. Select species: search 1 species at a time3. Assembly: the official backbone DNA sequence4. Position: location in the genome to examine5. Image width: how many pixels in display window; 5000 max6. Configure: make fonts bigger + other choices

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The Genome Browser Gatewaysample search for Human BRCA1

� Sample search: human, May 2004 assembly, BRCA1

•Often you will have to select the right gene from a results list

•Sometimes, you will go directly to a browser image (use an ID)

•AF005068, breast cancer 1, early onset

select


Overview of the wholeGenome Browser page

(first day, new human release)

}Genome viewer section

Track and image controls(day 1 = 40 tracks)


Overview of the wholeGenome Browser page

(mature release)

}Genome viewer section

mRNA and EST Tracks

Expression and Regulation

Comparative Genomics

ENCODE TracksVariation and Repeats

Groups of dataMapping and Sequencing Tracks

Genes and Gene Prediction Tracks


Different species, different tracks, same software


Sample Genome Viewer image, BRCA1 region

Genome backboneSTS markers

Known genes

RefSeq genes

Gene predictions

GenBank mRNAs

repeats

GenBank ESTs

conservation

SNPs

MGC clones


Annotation Track options, defined

� Hide: removes a track from view

� Dense: all items collapsed into a single line

� Squish: each item = separate line, but 50% height + packed

� Pack: each item separate, but efficiently stacked (full height)

� Full: each item on separate line


Clicking an annotation line, new page of detailed information

You will get detail for that single item you clickExample: click on the BRCA1 Black “Known Genes” line

Click the line

Newweb page

opens

Many details and links to more data about BRCA1


Click annotation track = BRCA1 “Known gene” detail page informative

description

other resource links

microarray data

mRNA secondary structure

links to sequences

protein domains/structure

homologs in other species

Gene Ontology™ descriptions

mRNA descriptions

pathways

Not all genes have This much detail.

Different annotation tracks

carry different detaildata.

SNP

detail page

sample


Getting the sequencesGet DNA, with Extended Options; or Details pages

� Use the DNA link at the top

� Plain or Extended options

� Change colors, fonts, etc.


Accessing the BLAT tool

� Rapid searches by INDEXING the entire genome� Works best with high similarity matches

BLAT = B LAST-l ike A lignment T ool


BLAT: Blast-like alignment tool

� Blat is really really fast—it has been optimized to search the whole genomes more quickly than BLAST does.

� UCSC have created an INDEX of all the unique 11mers if it’s DNA, 4mers if protein (or stretches of 11nucleotides or 4 amino acids).

� it looks down its index of 11mers, finds a match and works out from there.

� Blast does it the other way—it indexes your query and then runs your smaller index over everything…that’s the essential difference in the algorithm.


BLAT

� UCSC documentation:� “On DNA queries, BLAT is designed to quickly find

sequences with 95% or greater similarity of length 40 bases or more . It may miss genomic sequences that are more divergent or shorter than these minimums, although it will find perfect sequence matches of 33 bases and sometimes as few as 22 bases. The tool is capable of aligning sequences that contain large introns.

� On protein queries, BLAT rapidly locates genomic sequences with 80% or greater similarity of length 20 amino acids or more . In general, gene family members that arose within the last 350 million years can generally be detected.”


BLAT tool overview: www.openhelix.com/sampleseqs.html

Submit

� Make choices

DNA limit 25000 basesProtein limit 10000 aa25 total sequences

� Paste one or more

sequences

� Or upload


BLAT results, with links

� Results with demo sequences, settings default; sort = Query, Score� Score is a count of matches—higher number, better match

sorting

� Click browser to go to Genome Browser image location (next slide)� Click details to see the alignment to genomic sequence (2nd slide)


BLAT results, alignment details browser

� From browser click in BLAT results� A new line with your Sequence from BLAT Search appears!

query

matches

Click to flip frame

� Watch out for reading frame! Click - - - > to flip frame� Base position = full and zoomed in enough to see

amino acids


BLAT results,alignment details

Your query

Genomic match, color cues

Side-by-side alignment


In Silico PCR: find genomic sequence using primers

� Select genome� Enter primers� Minimum 15 bases� Flip reverse primer?� Submit

(note: the tool does not handle ambiguous bases at this time—don’t use Ns)


In Silico PCR: results

� Genomic location shown, links to Genome Viewer

location your primers

� Your primers displayed, flipped if necessary� Predicted genomic sequence shown

Tm for primers

� Primer melting temperatures provided

size

� Product size shown


Proteome Browser

� Access from homepage or Known Gene pages� Exon diagram, amino acids� Many protein properties (pI, mw, composition, 3D…)

moreprotein

data


Gene Sorter

� From homepage select ‘Gene sorter’


Gene Sorter interface

� Sorts genes by several criteria


Gene Sorter interface

� Choose from 11 sorting options


Gene Sorter results

GENOMICA - Unifem.docente.unife.it/.../genomica-1/Prokopenko_Lezione1.pdfIl Progetto Genoma Umano...

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