Bioinformatics Jack Min Office 3012 Office hours: TR 12:15 – 4

Bioinformatics

Jack MinOffice 3012Office hours: TR 12:15 – 4

How would you define "bioinformatics"?

Can you distinguish between a gene and a genome?

Do you know what a Blast search is?

Do you know what any of the letters in "blast" represent?

What is Genbank?What is NCBI?What is meant by "codon usage"?

Here are two nucleotide sequences:(i) AGTCGGTAACCTAAG(ii) GGCAAAUACUAAGGA

Which of these is a DNA sequence? Explain your answer.

Here is a DNA sequence: 5' ATTGGRTCCAATA 3‘

(i) What do the 5' and 3' mean?(ii) What does the R stand for?(iii) Write the sequence of the

complementary DNA strand, using the same notation.

What is meant by the "template" and "coding" strands of DNA?

Name three different kinds of RNA.Which amino acids are represented by the following symbols? A

LQKGW

International Union of Pure and Applied Chemistry

http://www.google.com/url?sa=t&source=web&cd=2&ved=0CCQQFjAB&url=http%3A%2F%2Fwww.iupac.org%2Fdhtml_home.html&ei=tzxsTJuQGYipnQfIncCxAg&usg=AFQjCNG8ZIa__I273npK1BfwRNv_9OCKxA

What is a PAM matrix?Can both nucleotide and amino acid sequences

be used to build molecular phylogenies?Explain the method of parsimony for building

sequence-based phylogenies.What is the program CLUSTAL used for?

What do you know about microarrays? For instance, are there different types of microarrays, and what are they used for?

This question is to test your general knowledge of genetics and molecular biology. Can you define the following terms?

(a) Gene

(b) Locus

(c) Allele

(d) Linkage

(e) Linkage disequilibrium

(f) synonymous substitution

(g) Intron

(h) concerted evolution

(i) Pleiotropy

(j) PCR

(k) RFLP

(l) Haplotype

• To provide an introduction to bioinformatics with a focus on the National Center for Biotechnology Information (NCBI) and EBI

• To focus on the analysis of DNA, RNA and proteins

• To introduce you to the analysis of genomes

• To combine theory and practice to help you solve research problems

What are the goals of the course?

Themes throughout the course

Textbooks

Web sites

Literature references

Gene/protein families

Computer labs

Textbook

Bioinformatics and Functional Genomics Second edition (Wiley, 2009).

ReferenceBioinformaticsThird edition (Wiley, 2005)Baxevanis and Ouellette

Web sites

The course website is reached via moodle: http://pevsnerlab.kennedykrieger.org/moodle(or Google “moodle bioinformatics”)--This site contains the powerpoints for each lecture. including color and black & white versions--The weekly quizzes are here

The textbook website is:http://www.bioinfbook.orgThis has powerpoints, URLs, etc. organized by chapter

Literature references

You are encouraged to read original source articles(posted on moodle). They will enhance your understanding of the material. Readings are optional but recommended.

http://ghr.nlm.nih.gov/handbook.pdfhttp://www.ncbi.nlm.nih.gov/books/NBK21101/

http://ghr.nlm.nih.gov/handbook.pdf

http://www.ncbi.nlm.nih.gov/books/NBK21101/

Themes throughout the course: gene/protein families

We will use beta globin and retinol-binding protein 4 (RBP4) as model genes/proteins throughout the course.

Globins including hemoglobin and myoglobin carry oxygen. RBP4 is a member of the lipocalin family. It is a small, abundant carrier protein. We will study globins and lipocalins in a variety of contexts including

--sequence alignment--gene expression--protein structure--phylogeny--homologs in various species

Computer labs

You can use any computer you can find –

Computer lab in the department, in your lab,

Computers in my lab (3013)

Outline for today

Definition of bioinformatics

Overview of the NCBI website

Accessing information about DNA and proteins--Definition of an accession number--Four ways to find information on proteins and DNA

Access to biomedical literature

Pairwise alignment: introduction

What is Bioinformatics?Broad definition:

The use of computational tools for:

acquiring,

storing and

analyzing

biological information.

Narrow definition:The use of computational tools for:

acquiring,storing andanalyzing

molecular sequence information.

• Interface of biology and computers

• Analysis of proteins, genes and genomes using computer algorithms and computer databases

• Genomics is the analysis of genomes. The tools of bioinformatics are used to make sense of the billions of base pairs of DNA that are sequenced by genomics projects.

What is bioinformatics?

Tool-users

Tool-makers

bioinformatics

public healthinformatics

medicalinformatics

infrastructure

databases algorithms

Three perspectives on bioinformatics

The cell

The organism

The tree of life

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DNA RNA phenotypeprotein

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Time ofdevelopment

Body region, physiology, pharmacology, pathology

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After Pace NR (1997) Science 276:734

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Fig. 2.1Page 17

Growth of GenBank + Whole Genome Shotgun(1982-November 2008)

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DNA RNA protein

Central dogma of molecular biology

genome transcriptome proteome

Central dogma of bioinformatics and genomics

DNA RNA

cDNAESTsUniGene

phenotype

genomicDNAdatabases

protein sequence databases

protein

Fig. 2.2Page 20

GenBankEMBL DDBJ

There are three major public DNA databases

The underlying raw DNA sequences are identical

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GenBankEMBL DDBJ

Housedat EBI

EuropeanBioinformatics

Institute

There are three major public DNA databases

Housed at NCBINational

Center forBiotechnology

Information

Housed in Japan

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The Trace Archive at NCBI containsover 2 billion traces

11/08

Taxonomy at NCBI:~200,000 species are represented in GenBank

http://www.ncbi.nlm.nih.gov/Taxonomy/txstat.cgi11/08

The most sequenced organisms in GenBank

Homo sapiens 13.1 billion basesMus musculus 8.4bRattus norvegicus 6.1bBos taurus 5.2bZea mays 4.6bSus scrofa (wild boar) 3.6bDanio rerio (zebrafish) 3.0bOryza sativa (japonica) 1.5bStrongylocentrotus purpurata (sea urchins) 1.4bNicotiana tabacum 1.1b

Updated 11-6-08GenBank release 168.0Excluding WGS, organelles, metagenomics

Table 2-2Page 18

National Center for BiotechnologyInformation (NCBI)

www.ncbi.nlm.nih.gov

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www.ncbi.nlm.nih.govFig. 2.5Page 25

Fig. 2.5Page 25

PubMed is… • National Library of Medicine's search service• 16 million citations in MEDLINE• links to participating online journals• PubMed tutorial (via “Education” on side bar)

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Entrez integrates…

• the scientific literature; • DNA and protein sequence databases; • 3D protein structure data; • population study data sets; • assemblies of complete genomes

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Entrez is a search and retrieval system that integrates NCBI databases

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BLAST is…

• Basic Local Alignment Search Tool• NCBI's sequence similarity search tool• supports analysis of DNA and protein databases• 100,000 searches per day

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OMIM is…

•Online Mendelian Inheritance in Man•catalog of human genes and genetic disorders•created by Dr. Victor McKusick; led by Dr. Ada Hamosh

at JHMI

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Books is…

• searchable resource of on-line books

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TaxBrowser is…

• browser for the major divisions of living organisms (archaea, bacteria, eukaryota, viruses)• taxonomy information such as genetic codes• molecular data on extinct organisms

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Structure site includes…

• Molecular Modelling Database (MMDB)

• biopolymer structures obtained from

the Protein Data Bank (PDB)• Cn3D (a 3D-structure viewer)• vector alignment search tool (VAST)

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Outline for today



Accessing information about DNA and proteins--Definition of an accession number--Five ways to find information on proteins and DNA



Accession numbers are labels for sequences

NCBI includes databases (such as GenBank) that contain information on DNA, RNA, or protein sequences. You may want to acquire information beginning with a query such as the name of a protein of interest, or theraw nucleotides comprising a DNA sequence of interest. DNA sequences and other molecular data are tagged with accession numbers that are used to identify a sequenceor other record relevant to molecular data.

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What is an accession number?

An accession number is label that used to identify a sequence. It is a string of letters and/or numbers that corresponds to a molecular sequence.

Examples (all for retinol-binding protein, RBP4):

X02775 GenBank genomic DNA sequenceNT_030059 Genomic contigRs7079946 dbSNP (single nucleotide polymorphism)

N91759.1 An expressed sequence tag (1 of 170)NM_006744 RefSeq DNA sequence (from a transcript)

NP_007635 RefSeq proteinAAC02945 GenBank proteinQ28369 SwissProt protein1KT7 Protein Data Bank structure record

protein

DNA

RNA

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Five ways to access DNA and protein sequences

[1] Entrez Gene with RefSeq[2] UniGene [3] European Bioinformatics Institute (EBI) and Ensembl (separate from NCBI)[4] ExPASy Sequence Retrieval System (separate from NCBI)[5] UCSC Genome Browser

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5 ways to access protein and DNA sequences

[1] Entrez Gene with RefSeq

Entrez Gene is a great starting point: it collectskey information on each gene/protein from major databases. It covers all major organisms.

RefSeq provides a curated, optimal accession number for each DNA (NM_006744) or protein (NP_007635)

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From the NCBI homepage, type “beta globin”and hit “Go”

revised 11/08Fig. 2.7Page 29

revisedFig. 2.7Page 29

By applying limits, there are now fewer entries

revisedFig. 2.8Page 30

Entrez Gene (top of page)

Note that links tomany other HBB database entries are available

Entrez Gene (middle of page)

Entrez Gene (middle of page, continued)

Entrez Gene (bottom of page): RefSeqs

Entrez Gene (bottom of page): non-RefSeq accessions

Fig. 2.9Page 32

Fig. 2.9Page 32

Fig. 2.9Page 32

FASTA format:versatile, compact with

>one header line followed by a string of nucleotides or amino acids in the single letter code

Fig. 2.10Page 32



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DNA RNA

complementary DNA(cDNA)

protein

UniGene

Fig. 2.3Page 23

UniGene: unique genes via ESTs

• Find UniGene at NCBI: www.ncbi.nlm.nih.gov/UniGene

• UniGene clusters contain many expressed sequence tags (ESTs), which are DNA sequences (typically 500 base pairs in length) corresponding to the mRNA from an expressed gene. ESTs are sequenced from a complementary DNA (cDNA) library.

• UniGene data come from many cDNA libraries. Thus, when you look up a gene in UniGene you get information on its abundance and its regional distribution.

Pages 20-21

Cluster sizes in UniGene

This is a gene with1 EST associated;the cluster size is 1 Fig. 2.3

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Cluster sizes in UniGene

This is a gene with10 ESTs associated;the cluster size is 10

Cluster sizes in UniGene (human)

Cluster size (ESTs) Number of clusters1 40,3002 18,5003-4 18,0005-8 13,4009-16 8,10017-32 5,200

500-1000 1,9001000-4000 9404000-16,000 7416,000-65,000 8

UniGene build 216, 11/0816000:70000[ESTC]

UniGene: unique genes via ESTs

Conclusion: UniGene is a useful tool to look upinformation about expressed genes. UniGenedisplays information about the abundance of atranscript (expressed gene), as well as its regionaldistribution of expression (e.g. brain vs. liver).

We will discuss UniGene further in gene expression section.

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Ensembl to access protein and DNA sequences

Try Ensembl at www.ensembl.org for a premierhuman genome web browser.

We will encounter Ensembl as we study the human genome,BLAST, and other topics.

clickhuman

enterRBP4



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ExPASy to access protein and DNA sequences

ExPASy sequence retrieval system(ExPASy = Expert Protein Analysis System)

Visit http://www.expasy.ch/

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Fig. 2.11Page 33



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[1] Visit http://genome.ucsc.edu/, click Genome Browser

[2] Choose organisms, enter query (beta globin), hit submit

Example of how to access sequence data:HIV-1 pol

There are many possible approaches. Begin at the mainpage of NCBI, and type an Entrez query: hiv-1 pol

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11/08

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Searching for HIV-1 pol:Following the “genome” link yields

a manageable five results

Example of how to access sequence data:HIV-1 pol

For the Entrez query: hiv-1 polthere are about 80,000 nucleotide or protein records(and >200,000 records for a search for “hiv-1”),but these can easily be reduced in two easy steps:

--specify the organism, e.g. hiv-1[organism]--limit the output to RefSeq!

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only 1 RefSeq

over 200,000nucleotide entriesfor HIV-1

Examples of how to access sequence data:histone

query for “histone” # results

protein records 21847RefSeq entries 7544

RefSeq (limit to human) 1108NOT deacetylase 697

At this point, select a reasonable candidate (e.g.histone 2, H4) and follow its link to Entrez Gene.There, you can confirm you have the right gene/protein.

8-12-06

Outline for today



Accessing information about DNA and proteins--Definition of an accession number--Four ways to find information on proteins and DNA



PubMed at NCBIto find literatureinformation

PubMed is the NCBI gateway to MEDLINE.

MEDLINE contains bibliographic citations and author abstracts from over 4,600 journals published in the United States and in 70 foreign countries.

It has >18 million records dating back to 1950s.

Page 35Updated 11-08

MeSH is the acronym for "Medical Subject Headings."

MeSH is the list of the vocabulary terms used for subject analysis of biomedical literature at NLM. MeSH vocabulary is used for indexing journal articles for MEDLINE.

The MeSH controlled vocabulary imposes uniformity and consistency to the indexing of biomedical literature.

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lipocalin AND disease(60 results)

lipocalin OR disease(1,650,000 results)

lipocalin NOT disease(530 results)

1 AND 2

1 OR 2

1 NOT 2

1

1

1

2

2

2

Fig. 2.12Page 34

true positive“globin” is found

“globin” is not found

“globin” is present

“globin” is absent

Article contents:

Search result:

true negativefalse negative(article discusses

globins)

false positive(article does not

discuss globins)

Pairwise sequence alignment

Outline: pairwise alignment

• Overview and examples• Definitions: homologs, paralogs, orthologs• Assigning scores to aligned amino acids: Dayhoff’s PAM matrices• Alignment algorithms: Needleman-Wunsch, Smith-Waterman• Statistical significance of pairwise alignments

• It is used to decide if two proteins (or genes) are related structurally or functionally

• It is used to identify domains or motifs that are shared between proteins

• It is the basis of BLAST searching

• It is used in the analysis of genomes

Pairwise sequence alignment is the most fundamental operation of bioinformatics

Pairwise alignment: protein sequencescan be more informative than DNA

• protein is more informative (20 vs 4 characters); many amino acids share related biophysical properties

• codons are degenerate: changes in the third position often do not alter the amino acid that is specified

• protein sequences offer a longer “look-back” time

• DNA sequences can be translated into protein, and then used in pairwise alignments

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• DNA can be translated into six potential proteins

5’ CAT CAA 5’ ATC AAC 5’ TCA ACT

5’ GTG GGT 5’ TGG GTA 5’ GGG TAG


5’ CATCAACTACAACTCCAAAGACACCCTTACACATCAACAAACCTACCCAC 3’3’ GTAGTTGATGTTGAGGTTTCTGTGGGAATGTGTAGTTGTTTGGATGGGTG 5’


• Many times, DNA alignments are appropriate--to confirm the identity of a cDNA--to study noncoding regions of DNA--to study DNA polymorphisms--example: Neanderthal vs modern human DNA

Query: 181 catcaactacaactccaaagacacccttacacccactaggatatcaacaaacctacccac 240 |||||||| |||| |||||| ||||| | ||||||||||||||||||||||||||||||| Sbjct: 189 catcaactgcaaccccaaagccacccct-cacccactaggatatcaacaaacctacccac 247

Pairwise alignment The process of lining up two sequences to achieve maximal levels of identity (and conservation, in the case of amino acid sequences) for the purpose of assessing the degree of similarity and the possibility of homology.

Definitions

HomologySimilarity attributed to descent from a common ancestor.

Definitions

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HomologySimilarity attributed to descent from a common ancestor.

Definitions

Identity

The extent to which two (nucleotide or amino acid) sequences are invariant.

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RBP: 26 RVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVA 59 + K++ + ++ GTW++MA + L + A glycodelin: 23 QTKQDLELPKLAGTWHSMAMA-TNNISLMATLKA 55

Orthologs Homologous sequences in different species that arose from a common ancestral gene during speciation; may or may not be responsible for a similar function.

Paralogs Homologous sequences within a single species that arose by gene duplication.

Definitions: two types of homology

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Orthologs:members of a gene (protein)family in variousorganisms.This tree showsRBP orthologs.

common carp

zebrafish

rainbow trout

teleost

African clawed frog

chicken

mouserat

rabbitcowpighorse

human

10 changes Page 43

Paralogs:members of a gene (protein)family within aspecies

apolipoprotein D

retinol-bindingprotein 4

Complementcomponent 8

prostaglandinD2 synthase

neutrophilgelatinase-associatedlipocalin

10 changesLipocalin 1Odorant-bindingprotein 2A

progestagen-associatedendometrialprotein

Alpha-1Microglobulin/bikunin

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1 MKWVWALLLLAAWAAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEG 50 RBP . ||| | . |. . . | : .||||.:| : 1 ...MKCLLLALALTCGAQALIVT..QTMKGLDIQKVAGTWYSLAMAASD. 44 lactoglobulin

51 LFLQDNIVAEFSVDETGQMSATAKGRVR.LLNNWD..VCADMVGTFTDTE 97 RBP : | | | | :: | .| . || |: || |. 45 ISLLDAQSAPLRV.YVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTK 93 lactoglobulin

98 DPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAV...........QYSC 136 RBP || ||. | :.|||| | . .| 94 IPAVFKIDALNENKVL........VLDTDYKKYLLFCMENSAEPEQSLAC 135 lactoglobulin

137 RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQ.EELCLARQYRLIV 185 RBP . | | | : || . | || | 136 QCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI....... 178 lactoglobulin

Pairwise alignment of retinol-binding protein 4 and -lactoglobulin

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SimilarityThe extent to which nucleotide or protein sequences are related. It is based upon identity plus conservation.

IdentityThe extent to which two sequences are invariant.

Conservation Changes at a specific position of an amino acid or (less commonly, DNA) sequence that preserve the physico-chemical properties of the original residue.

Definitions





Pairwise alignment of retinol-binding protein and -lactoglobulin

Identity(bar)

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Somewhatsimilar

(one dot)

Verysimilar

(two dots)

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Internalgap

Terminalgap

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• Positions at which a letter is paired with a null are called gaps.

• Gap scores are typically negative.

• Since a single mutational event may cause the insertion or deletion of more than one residue, the presence of a gap is ascribed more significance than the length of the gap.

• In BLAST, it is rarely necessary to change gap values from the default.

Gaps

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Bioinformatics Jack Min Office 3012 Office hours: TR 12:15 – 4