Genomics and Personalized Care

Lab Session

Leming Zhou, PhDSchool of Health and Rehabilitation

SciencesDepartment of Health Information

Management

Outline• Nucleotide, protein, genetic variation,

gene and disease association databases– NCBI

• GenBank; protein structure; dbSNP; OMIM

• Pairwise sequence alignment• BLAST search• UCSC genome browser

NCBI• Created as a part of National Library of

Medicine in 1988– Establish public databases

– Perform research in computational biology

– Develop software tools for sequence analysis

– Disseminate biomedical information

• Databases– Sequence, such as GeneBank, RefSeq, dbSNP

– Literature, such as PubMed, OMIM

• Tools– Entrez. Blast, Cn3D, etc.

NCBI Homepage

GenBank• Nucleotide only sequence database

• GenBank Data– Direct submissions individual records (BankIt, Sequin)

– Batch submissions via email (EST, GSS, STS)

– ftp accounts established for sequencing centers

• Data shared nightly amongst three collaborating databases:– GenBank

– DNA Database of Japan (DDBJ).

– European Molecular Biology Laboratory Database (EMBL)

GenBank Record (Header)LOCUS NM_001963 4913 bp mRNA linear PRI 20-SEP-2009

DEFINITION Homo sapiens epidermal growth factor (beta-urogastrone) (EGF), mRNA.

ACCESSION NM_001963

VERSION NM_001963.3 GI:166362727

KEYWORDS .

SOURCE Homo sapiens (human)

ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo.

REFERENCE 1 (bases 1 to 4913)

AUTHORS Hosgood,H.D. III, Menashe,I., He,X., Chanock,S. and Lan,Q.

TITLE PTEN identified as important risk factor of chronic obstructive pulmonary disease

JOURNAL Respir Med (2009) In press

PUBMED 19625176

REMAKR GeneRIF: Observational study of gene-disease association.

GenBank Record (Features)FEATURES Location/Qualifiers

source 1..4913 /organism="Homo sapiens" /mol_type="mRNA" /db_xref="taxon:9606" /chromosome="4" /map="4q25"

gene 1..4913 /gene="EGF" /gene_synonym="HOMG4; URG" /note="epidermal growth factor (beta-urogastrone)" /db_xref="GeneID:1950" /db_xref="HGNC:3229" /db_xref="HPRD:00578" /db_xref="MIM:131530"

exon 1..579 /gene="EGF" /gene_synonym="HOMG4; URG" /inference="alignment:Splign" /number=1

CDS 453..4076 /gene="EGF" /gene_synonym="HOMG4; URG" /note="beta-urogastrone" /codon_start=1 /product="epidermal growth factor precursor" /protein_id="NP_001954.2" /db_xref="GI:166362728" /db_xref="CCDS:CCDS3689.1" /db_xref="GeneID:1950" /db_xref="HGNC:3229" /db_xref="HPRD:00578" /db_xref="MIM:131530" /translation="MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGP APFLIFSHGNSIFRIDTEGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVF

GenBank Record (Sequence)ORIGIN

1 aaaaagagaa actgttggga gaggaatcgt atctccatat ttcttctttc agccccaatc

61 caagggttgt agctggaact ttccatcagt tcttcctttc tttttcctct ctaagccttt

121 gccttgctct gtcacagtga agtcagccag agcagggctg ttaaactctg tgaaatttgt

181 cataagggtg tcaggtattt cttactggct tccaaagaaa catagataaa gaaatctttc

241 ctgtggcttc ccttggcagg ctgcattcag aaggtctctc agttgaagaa agagcttgga

301 ggacaacagc acaacaggag agtaaaagat gccccagggc tgaggcctcc gctcaggcag

361 ccgcatctgg ggtcaatcat actcaccttg cccgggccat gctccagcaa aatcaagctg

421 ttttcttttg aaagttcaaa ctcatcaaga ttatgctgct cactcttatc attctgttgc

481 cagtagtttc aaaatttagt tttgttagtc tctcagcacc gcagcactgg agctgtcctg

541 aaggtactct cgcaggaaat gggaattcta cttgtgtggg tcctgcaccc ttcttaattt

601 tctcccatgg aaatagtatc tttaggattg acacagaagg aaccaattat gagcaattgg

661 tggtggatgc tggtgtctca gtgatcatgg attttcatta taatgagaaa agaatctatt

721 gggtggattt agaaagacaa cttttgcaaa gagtttttct gaatgggtca aggcaagaga

781 gagtatgtaa tatagagaaa aatgtttctg gaatggcaat aaattggata aatgaagaag

841 ttatttggtc aaatcaacag gaaggaatca ttacagtaac agatatgaaa ggaaataatt

901 cccacattct tttaagtgct ttaaaatatc ctgcaaatgt agcagttgat ccagtagaaa

961 ggtttatatt ttggtcttca gaggtggctg gaagccttta tagagcagat ctcgatggtg

FASTA: Sequence Format

Protein Structure

Crystal Structure of a Protein

Protein Structure Databases• Proteins take on 3D structure

• 3D data for some proteins is available due to techniques such as NMR and X-Ray crystallography– PDB http://www.pdb.org/

– SCOP http://scop.mrc-lmb.cam.ac.uk/scop

– MMDB http://www.ncbi.nlm.nih.gov/Structure/

Genetic Variations

Polymorphisms• Genomic sequences from two unrelated

individuals are 99.9% identical.

• The 0.1% difference is due to genetic variations, and mainly (~90%) one form of variation called Single Nucleotide Polymorphisms (SNPs, single-base variations).

Importance of Genetic Variations• Genetic variations underlie phenotypic

differences among different individuals

• Genetic variations determine our predisposition to diseases and responses to drugs, therapies, and environmental insults such as bacteria, virus, and chemicals

• Genetic variations reveal clues of ancestral human migration history

Major Types of Genetic Variations• Single nucleotide mutation

– Majority of SNPs do NOT directly contribute to any phenotypes

• Insertion or deletion of one or more nucleotides– Tandem repeat polymorphisms (Genomic regions consisting of

variable length, usually 1-100 bases long, of sequence motifs repeating in tandem with variable copy number)

• Used as genetic markers for DNA finger printing (forensic, parentage testing)

• Many cause genetic diseases

– Insertion/Deletion polymorphisms (Often resulted from localized rearrangements between homologous tandem repeats)

• Gross chromosomal aberration– Deletions, inversions, or translocation of large DNA fragments

– Often causing serious genetic diseases

The Effect of SNPs• The phenotypic consequence of a SNP is

significantly affected by the location where it occurs (gene or non-gene), as well as the nature of the mutation (synonymous or non-synonymous)– No consequence

– Affect gene transcription quantitatively or qualitatively

– Affect gene translation quantitatively or qualitatively

– Change protein structure and functions

– Change gene regulation at different steps

Simple/Complex Genetic Diseases and SNPs• Simple genetic diseases (Mendelian diseases) are

often caused by mutations in a single gene– e.g. Huntington’s, Cystic fibrosis, etc.

• Many complex diseases are the result of mutations in multiple genes, the interactions among them as well as between the environmental factors– e.g. cancers, heart diseases, Alzheimer's, diabetes,

asthmas, obesity, etc.

Genetic Variations Databases• dbSNP

– http://www.ncbi.nlm.nih.gov/SNP/

• Online Mendelian Inheritance in Man (OMIM)– http://www.ncbi.nlm.nih.gov/omim

• International HapMap Project– http://www.hapmap.org/

• Genome Variation Server (Seattle SNPs)– http://gvs.gs.washington.edu/GVS/

dbSNP• The Single Nucleotide Polymorphism database (dbSNP) is

a public- domain archive for a broad collection of simple genetic variations

• This collection of polymorphisms includes:– Single-base nucleotide substitutions (or single nucleotide

polymorphisms -SNPs)

• Roughly 10 million in human population or on average 1 per 300 bps

• Less than half of these SNPs are identified and stored in the database

– Microsatellite repeat variations (or short tandem repeats - STRs)

• In sillico estimation of potentially polymorphic variable number tandem repeats (VNTR) are over 100,000 across the human genome

– Small-scale multi-base deletions or insertions

• The short insertion/deletions are difficult to quantify and the number is likely to fall in between SNPs and VNTR

A dbSNP Record>gnl|dbSNP|ss5586300|allelePos=214|len=475|

taxid=9606|alleles='A/G'|mol=Genomic ATAAACATGG ACTTTTACAA AACCCATATC GTATACCACC ACTTTTTCCCATCAAGTCAT YTGTTAAAAC TAAATGTAAG AAAAATCTGC TAGAGGAAAACTTTGAGGAA CATTCAATRT CACCTGAAAG AGAAATGGGA AATGAGAACATTCCAAGTAC AGTGAGCACA ATTAGCCGTA ATAACATTAG AGAAAATGTT TTTAAAGRAG CCA R CTCAAGCAAT ATTAATGAAG TAGGTTCCAG TACTAATGAA GTGGGCTCCAGTATTAATGA AATAGGTTCC AGTGATGAAA ACATTCAAGC AGAACTAGGT AGAAACAGAG GGCCAAAATT GAATGCTATG CTTAGATTAG GGGTTTTGCA ACCTGAGGTC TATAAACAAA GTCTTCCTGG AAGTAATTGT AAGCATCCTGAAATAAAAAA GCAAGAATAT GAAGAAGTAG TTCAGACTGT TAATACAGAT TTCTCTCCAT A

Different Ways to Search SNPs in dbSNP• dbSNP web site

– Direct search of SS record; batch search; allow SNP record submission; No search limit

• Entrez SNP

– http://www.ncbi.nlm.nih.gov/sites/entrez?db=Snp

– Search limits options allows precise retrieval

• Entrez Gene Record’s SNP Links Out Feature

– Direct links to corresponding SNP records; access to genotype and linkage disequilibrium data

• NCBI’s MapViewer

– Visualize SNPs in the genomic context along with other types of genetic data

Search SNPs from dbSNP Web Page • http://www.ncbi.nlm.nih.gov/SNP/index.html

Search SNPs from Entrez SNP Web Page• http://www.ncbi.nlm.nih.gov/sites/entrez?db=Snp

• The dbSNP is a part of the Entrez integrated information retrieval system and may be searched using either qualifiers or a combination search limits from 14 different categories

Gene and Disease

Disease Causing GenesDisease centric databases:

• OMIM: http://www.ncbi.nlm.nih.gov/omim/

• CDC HugeNavigator: http://hugenavigator.net/

• HGMD: https://portal.biobase-international.com/hgmd/pro/start.php

• A Catalog of Published Genome-Wide Association Studies: http://www.genome.gov/26525384

Online Mendelian Inheritance in Man (OMIM)• http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM • OMIM is a human genetic disorders database built and curated

using results from published studies

• Each OMIM record provides a summary of the current state of knowledge of the genetic basis of a disorder, which contains the following information:

– description and clinical features of a disorder or a gene involved in genetic disorders;

– biochemical and other features;

– cytogenetics and mapping;

– molecular and population genetics;

– diagnosis and clinical management;

– animal models for the disorder;

– allelic variants.

• OMIM is searchable via NCBI Entrez, and its records are cross-linked to other NCBI resources.

OMIM: Variant• The OMIM database includes genetic disorders

caused by various mutation/variation, from SNPs to large-scale chromosomal abnormalities

• Variants are represented by a 10-digit OMIM number, and can be searched in two ways– Search for a gene or a disease, when retrieved, view its

variants

Variants in OMIM Records• For most genes, only selected mutations are

included – Criteria for inclusion include: the first mutation to be discovered, high

population frequency, distinctive phenotype, historic significance, unusual mechanism of mutation, unusual pathogenetic mechanism, and distinctive inheritance.

• Most of the variants represent disease-producing mutations, NOT polymorphisms.

• A few polymorphisms are included, many of which show a positive statistical correlation with particular common disorders.

• Few neutral polymorphisms are included in OMIM

• Some SNPs in the dbSNP records are not linked to the corresponding OMIM records.

Similarity Search• Find statistically significant matches to a

protein or DNA sequence of interest. • Obtain information on inferred function of

the gene• Sequence identity/similarity is a

quantitative measurement of the number of nucleotides / amino acids which are identical /similar in two aligned sequences– Calculated from a sequence alignment

– Can be expressed as a percentage

– In proteins, some residues are chemically similar but not identical

Sequence Alignment• A linear, one-to-one correspondence

between some of the symbols in one sequence with some of the symbols in another sequence– Four possible outcomes in aligning two

sequences

• Identity; mismatch; gap in one sequence; gap in the other sequence

• May be DNA or protein sequences.

Alignment Algorithms• Sequences often contain highly conserved

regions

• These regions can be used for an initial alignment

Alignments• Two sequencesSeq 1: ACGGACTSeq 2: ATCGGATCT• There may be multiple ways of creating

the alignment. Which alignment is the best?

A – C – G G – A C T| | | | |A T C G G A T - C T

 A T C G G A T C T| | | | | |A – C G G – A C T

BLAST• BLAST - Basic Local Alignment Search Tool: A

sequence comparison algorithm optimized for speed used to search sequence databases for optimal local alignments to a query

• Most widely used and referenced computational biology resource

• The central idea of the BLAST algorithm is to confine attention to segment pairs that contain a word pair of length W with a score of at least T when compared to the query using a substitution matrix

• Word hits are then extended in both directions to generate an alignment with score exceeding a given threshold S

Four Steps of a BLAST search• Enter query sequence

• Select one BLAST program

• Choose the database to search

• Set optional parameters

Enter Query Sequence• Sequence can be pasted into a text field in FASTA

format or as accession number• Sequence can also be uploaded as a file (FASTA

format)• Users may indicate a sequence range of the query

sequence instead of using the whole query sequence• Job title will be automatically generated from

sequence header

Select one BLAST Program• BLAST Programs:

– BLASTN: DNA query sequence against a DNA database

– BLASTP: protein query sequence against a protein database

– BLASTX: DNA query sequence, translated into all six reading frames, against a protein database

– TBLASTN: protein query sequence against a DNA database, translated into all six reading frames

– TBLASTX: DNA query sequence, translated into all six reading frames, against a DNA database, translated into all six reading frames

• Choose the right one according to the purpose of the search

Choose the Database to Search• BLASTN

page 93

Optional Parameters• Specify the organism to search or exclude

– Common name, taxonomy id, …• Exclude certain sequences

– Exclude predicted sequences or sequences from metagenomics

• Use Entrez query to select a subset of the blast database

BLASTN Output (header)

BLASTN Output (Graphic Summary)

matches to itself

probable homologs

distantly related

homologs

distant homolog with shared

domain or motif

BLASTN Output (Descriptions)

BLASTN Output (Sequence Alignments)

Genome Browser

• Genome Browser is a computer program which helps to display gene maps, browse the chromosomes, align genes or gene models with ESTs or contigs etc.

• Big Three:– UCSC Genome Browser

– NCBI Mapviewer

– Ensemble

UCSC Genome Browser• http://genome.ucsc.edu

Organization of Genomic Data

Genome backbone: base position numbersequenceA

nnot

atio

n T

rack

s

chromosome band

known genes

predicted genes

evolutionary conservation

SNPs

sts sites

microarray/expression data

repeated regions

more…

Links out to more data

UCSC Genome Browser

UCSC Genome BrowserA

nno

tatio

n T

rack

s

sequenceSTS sites

Known gene

SNP

Evolutionary conservation

Repeated regions

Expression

A Sample of the UCSC Genome Browser

gene details

An

nota

tion

Tra

cks

sequence

comparisons

SNPs

Genome Browser Gateway

• Use this Gateway to search by:– Gene names, symbols, IDs

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

– Keywords: kinase, receptor

• See lower part of page for help with format

Genome Browser Gateway

Helpful search examples

samples provided

text/ID searches

The Genome Browser Gateway

Make your Gateway choices:1. Select Clade2. Select genome = 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

4 51 32

assembly

6

Different Species, Different Tracks

• Species may have different data tracks

• Layout, software, functions are the same

Sample Genome Viewer Image, TP53

base position

UCSC genes

RefSeq genes

mRNAs & ESTs

repeats

many species compared

SNPs

single species compared

MGC clones

Visual Cues on the Genome Browser

Track colors may have meaning—for example, UCSC Gene track:

•If there is a corresponding PDB entry = black•If there is a corresponding reviewed/validated seq = dark blue•If there is a non-RefSeq seq = lightest blue

Tick marks; a single location (STS, SNP)

For some tracks, the height of a bar is increased likelihood of an evolutionary relationship (conservation track)

Intron and direction of transcription <<< or >>>

<exon exon exon< < < < < < <ex 5' UTR3' UTR

Alignment indications (Conservation pairs: “chain” or “net” style)•Alignments = boxes, Gaps = lines

Options for Changing Images: Upper Section

• Change your view or location with controls at the top

• Use “base” to get right down to the nucleotides

• Configure: to change font, window size, more…– Next item, next exon navigation assistance can be turned on

Specifya

position

Fonts,window,

next item,more

Walkleft orright

Zoomin

Zoomout

Click tozoom 3x

and re-center

Annotation Track Display Options

• Some data is ON or OFF by default

• Menu links to info about the tracks: content, methods

• You change the view with pulldown menus

• After making changes, REFRESH to enforce the change

enforcechange

s

Enforcechanges

Change track view

Links to infoand/or filters

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

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

Full: each item on separate line

Mid-page Options to Change Settings

• You control the views

• Use pulldown menus

• Configure options page

Reset, back to defaults Start from

scratch

Enforce any changes (hide, full, squish…)

Flip display to Genomic 3’5’

Base Level and Protein Sequences