Data Data Acquisition Acquisition

Tools & Tools & TechniquesTechniques

In this presentation……

Part 1 – Sequencing Technology

Part 2 – Genomic Databases

Part

1

Sequencing Sequencing TechnologyTechnology

Principles of DNA sequencing

• DNA sequencing is performed using an automated version of the chain termination reaction, in which limiting amounts of dideoxyribonucleotides generate nested sets of DNA fragments with specific terminal bases

• Four reactions are set up, one for each of the four bases in DNA, each incorporating a different fluorescent label

• The DNA fragments are separated by PAGE and the sequence is read by a scanner as each fragment moves to the bottom of the gel

Types of DNA sequencing

DNA sequences come in three major forms• Genomic DNA comes directly from the genome and

includes extragenic material as well as genes. In eukaryotes, genomic DNA contains introns

• cDNA is reverse-transcribed from mRNA and corresponds only to the expressed parts of the genome. It does not contain introns

• Recombinant DNA comes from the laboratory and comprises artificial DNA molecules such as cloning vectors

Genome sequencing strategies

Only short DNA molecules (~800 bp) can be sequenced in one read, so large DNA molecules, such as genomes, must first be broken into fragments. Genome sequencing can be approached in two ways

• Shotgun sequencing involves the generation of random DNA fragments, which are sequenced in large numbers to provide genome-wide coverage

• Clone contig sequencing involves the systematic production and sequencing of subclones

Sequence quality control

• High quality sequence data is generated by performing multiple reads on both DNA strands

• Preliminary trace data is then base called and assessed for quality using a program such as Phred

• Vector sequences and repeated DNA elements are masked off and then the sequence is assembled into contigs using a program such as Phrap

• Remaining inconsistencies must be addressed by human curators

Single-pass sequencing

• Sequence data of lower quality can be generated by single reads (single-pass sequencing)

• Although somewhat inaccurate, single-pass sequences such as ESTs and GSSs can be generated in large amounts very quickly and inexpensively

RNA sequencing

Most RNA sequencing are deduced from the corresponding DNA sequences but special methods are required for the identification of modified nucleotides. These include biochemical assays, NMR spectroscopy and MS

Protein sequencing• Most protein sequencing is now-a-days carried out by

MS, a technique in which accurate molecular masses are calculated from the mass/charge ration of ions in a vacuum

• Soft ionization methods allow MS analysis of large macromolecules such as proteins

• Sequences can be deduced by comparing the masses of tryptic peptide fragments to those predicted from virtual digests of proteins in databases

• Also, de novo sequencing can be carried out by generating nested sets of peptide fragments in a collision cell and calculating difference in mass between fragments differing in length by a single amino acid residue

Importance of protein interactions• They underlie most cellular functions. Protein-protein

interactions result in formation of transient or stable multi-subunit complexes

• Understanding of these complexes is required for functional annotation of proteins and is a step towards the elucidation of molecular pathways such as signaling cascades and regulatory networks

• Protein interactions with nucleic acids form an important area of study, since such interactions are required for replication, transcription, recombination, DNA repair and many other processes. Proteins also interact with small molecules, which act as ligands, substrates, cofactors and allosteric regulators

Methods for protein interactions

• Genetic methods– Suppressor mutant

– Synthetic lethal effect

– Dominant negative mutations

• Affinity methods– Affinity chromatography

– Co-immunoprecipitation

• Molecular and atomic methods– X-ray crystallography– NMR spectroscopy– Other methods

• FRET• SPR spectroscopy• SELDI

• Library-based methods– Y2H system

Other methods

• For larger proteins that do not readily form crystals, alternative analytical methods are required to deduce structures

• These include X-ray fiber diffraction, electron microscopy and circular dichroism (CD) spectroscopy

Protein structure determination

• X-ray crystallography

• NMR spectroscopy

• Other methods– X-ray fiber diffraction– Electron microscopy– CD spectroscopy

X-ray crystallography

• Involves determination of protein structure by studying diffraction pattern of X-rays through a precisely orientated protein crystal

• They way in which X-rays are scattered depends on the electron density and spatial orientation of the atoms in the crystal

• A mathematical method called the Fourier transform is used to reconstruct electron density maps from the diffraction data allowing structural models to be built

NMR spectroscopy• NMR is a property of certain atoms that can switch

between magnetic states in an applied magnetic field by absorbing electromagnetic radiation

• The nature of absorbance spectrum is influenced by the type of atom and its chemical context, so that NMR spectroscopy can discriminate between different chemical groups

• NMR spectra are also modified by the proximity of atoms in space

• Analysis of NMR spectra allows 3D configuration of atoms to be reconstructed, resulting in a series of structural models

• The technique is suitable only for the analysis of small, soluble proteins

2-D gel electrophoresis

• The current method for studying proteins consists in part of a technique called two dimensional gel electrophoresis, which separates proteins by charge and size

• In the technique, researchers squirt a solution of cell contents onto a narrow polymer strip that has a gradient of acidity. When the strip is exposed to an electric current, each protein in the mixture settles into a layer according to its charge. Next, the strip is placed along the edge of a flat gel and exposed to electricity again. As the proteins migrate through the gel, they separate according to their molecular weight. What results is a smudgy patterns of dots, each of which contains a different protein

• In academic laboratories, scientists generally use a tool similar to a hole puncher to cut the protein spots from 2-D gels for individual identification by another method, mass spectroscopy

• Now-a-days, companies have started using robots to do it

Part

2

Genomic Genomic DatabasesDatabases

Types of databases

• There are many types of databases available for researchers in the field of biology– Primary sequence databases - for storage of raw

experimental data– Secondary databases - contain information on

sequence patterns and motifs– Organism specific databases– Other databases

Primary sequence databases

• Three primary sequence databases are GenBank (NCBI), the Nucleotide Sequence Database (EMBL) and the DNA Databank of Japan (DDBJ)

• These are repositories for raw sequence data, but each entry is extensively annotated and has features table to highlight the important properties of each sequence

• The three databases exchange data on a daily basis

Subsidiary sequence databases

• Particular types of sequence data are stored in subsidiaries of the main sequence databases. For instance, ESTs are stored in dbEST, a division of GenBank

• There are also subsidiary databases for GSSs and unfinished genomic sequence data

Organism specific resource

• As well as general databases that serve the entire biology community, there are many organism specific databases that provide information and resources for those researches working on particular species

• The number of such databases is growing as more genome projects are initiated, and many can be accessed from general genomics gateway sites such as GOLD

Organism-specific genomic databasesOrganism Database/resource URL

Escherichia coli EcoGene

EcoCyc (Encyclopedia of E. coli genes and metabolism

Colibri

http://bmb.med.miami.edu/EcoGene/EcoWeb

http://ecocyc.pangeasystems.com/ecocyc/ecocyc.html

http://genolist.pasteur.fr/Colibri

Bacillus subtilis SubtiList http://genolist.pasteur.fr/SubtiList

Saccharomyces cerevisiae

Saccharomyces Genome Database (SGD)

http://genome-www.stanford.edu/Saccharmyces

Plasmodium falciparum PlasmoDB http://PlasmoDB.org

Arabidopsis thaliana MIPS Arabidopsis thaliana Database (MAtDB)

The Arabidopsis information resource (TAIR)

http://mips.gsf.de/proj/thal/db

http://www.arabidopsis.org

Drosophila melanogaster

FlyBase http://flybase.bio.indiana.edu

Caenorhabditis elegans A C. elegans DataBase (ACeDB) http://www.acedb.org

Mouse Mouse Genome Database (MGD) http://www.informatics.jax.org

Human OnLine Mendelian Inheritance in Man (OMIM)

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

Finding organism-specific databases

• Organism specific databases are widely distributed on the Internet

• In order to find and interrogate databases on specific organisms, it is necessary to use a gateway site to access relevant databases and information resources

• Worked examples are provided, using GOLD as the gateway and illustrated with Ebola virus, the bacterium E. coli, the fruit fly Drosophila melanogaster and the human genome

Useful gateway sites providing information on multiple, organism and genomic resources

Gateway site URLNCBI Genomic Biology www.ncbi.nlm.nih.gov/Genomes/

GOLD (Genomes OnLine Database)

wit.integratedgenomics.com/GOLD

Organism specific genomic databases

www.unl.edu/stc-95/ResTools/biotools/biotools10.html

TIGR Microbial Database www.tigr.org/tdb/mdb/mdbcomplete.html

Bacterial genomes genolist.pasteur.fr

Yeast database genome-www.stanford.edu/Saccharomyces/yeast_info.html

EnsEMBL genome database project www.ensembl.org

MIPS (Munich Information Centre for Protein Sequences)

mips.gsf.de

Nematode

Baker’s Yeast Cells

Other databases• Specialized sequence databases – for storage and analysis of

particular types of sequences e.g., rRNA and tRNA, introns, promoters and other regulatory elements

• OMIM – for study of human genetics and molecular biology• Incyte and UniGene – for providing gene sequences and

transcripts with expert annotation for use in drug design and research

• Structural databases – for protein structural data (e.g. PDB, MMDB) – containing X-ray Crys. and NMR studies

• Proteins and higher order functions – to store information on particular types of proteins such as receptors, signal transduction components, regulatory hierarchies and enzymes

• Literature databases – to store scientific articles with text search facility (e.g. Medline and PubMED)

Database tools for displaying and annotating genomic sequence data

Viewer format URL

Artemis www.sanger.ac.uk/Software/Artemis

ACeDB www.acedb.org/Tutorial/brief-tutorial/shtml

Apollo www.ensembl.org/apollo

EnsEMBL www.ensembl.org

NCBI map viewer www.ncbi.nlm.nih.gov

GoldenPath genome.ucsc.edu

Database formats

• There is no universally agreed format for genome databases and several viewers and browsers have been developed with graphical displays for genomic sequence analysis and annotation

• One of the most versatile formats is ACeDN (originally designed for the nematode C. elegans), which has an object-oriented database architecture and is now used in many applications outside the field of genomic bioinformatics

Common formats

• There are several conventions for representing nucleic acid and protein sequences, of which the following are widely used– NBRF/PIR– FASTA– GDE

• These formats have limited facilities for comments, which must include a unique identifier code and sequence accession number

Formats for multiple sequence alignment

• There are separate formats for multiple sequence alignment representation, of which the following are popular– MSF

– PHYLIP

– ALN

Files of structural data

• Structural data are maintained as flat files using the PDB format

• Such files contain orthogonal atomic co-ordinates together with annotations, comments and experimental details

Submission of sequences

• Sequences may be submitted to any of the three primary databases using the tools provided by the database curators

• Such tools include WebIn and BankIt, which can be used over the Internet, and Sequin, a stand-alone application

Database interrogation

• All the databases discussed above can be searched by sequence similarity

• However, detailed text-based searches of the annotations are also possible using tools such as Entrez

• The simplest way to cross-reference between the primary nucleotide sequence databases and SWISS-PROT is to search by accession number, as this provides an unambiguous identifier of genes and their products

Databases covered by EntrezCategory Database

Nucleic acid sequences Entrez nucleotides: sequences obtained from GenBank, RefSeq and PDB

Protein sequences Entrez protein: sequences obtained from SWISS-PROT, PIR, PRF, PDB and translations from annotated coding regions in GenBank and RefSeq

3D structures Entrez Molecular Modeling Database (MMDB)

Genomes Complete genome assemblies from many sources

PopSet From GenBank, set of DNA sequences that have been collected to analyze the evolutionary relatedness of a population

OMIM OnLine Mendelian Inheritance in Man

Taxonomy NCBI Taxonomy Database

Books Bookshelf

ProbeSet Gene Expression Omnibus (GEO)

3D domains Domains from the Entrez MMDB

Literature PubMED

Databases covered by DBGET/LinkDBCategory Database

Nucleic acid sequences GenBank, EMBL

Protein sequences SWISS-PROT, PIR, PRF, PDBSTR

3D structures PDB

Sequence motifs PROSITE, EPD, TRANSFAC

Enzyme reactions LIGAND

Metabolic pathways PATHWAY

Amino acid mutations PMD

Amino acid indices AAindex

Genetic diseases OMIM

Literature LITDB Medline

Organism-specific gene catalogs

E. coli, H. influenzae, M. genitalium, M. pneumoniae, M. jannashii, Synechocystis, S. cerevisiae