Expensive to make a lot of PCR primers
but the price of primer synthesis keeps dropping & there is an economy of scale
Assembly problems are minimized since both the order and the amount of overlap of reads are known
Shotgun SequencingShotgun sequencing takes maximum advantage of the speed and low cost of automated sequencing
relies totally on software to assembly a jumble of essentially random sequence reads into a coherent and accurate contig
TIGR demonstrated “proof of concept” on the genomes of Haemophilus influenzae, Methanococcus jannaschii, and Mycoplasma genitalium
Celera Genomics demonstrated the ability to shotgun sequence the entire human genome (?)
Human Genome AssemblyThe HGP vs. Celera race to sequence the entire human genome was a classic battle of different strategies
The HGP used an ordered cloning approach
Breaking the genome into mapped BAC clones, then shotgun sequencing the BACs
Celera used a modified shotgun method
Random clones of various sizes (size selected libraries)
Plus relative mapping of clone ends (they must be located in the assembly at the correct distance and orientations
Created custom software to handle the assembly
Celera did make use of the “scaffold” built by the HGP
Other Large Sequencing ProjectsPhylogenetic identification/analysis
medical studies of bacteria
environmental samples
EST sequencing - differential expression
cDNA studies
alternate splicing
full length transcripts
Genotyping
score known alleles
identify new mutations
AutomationThe "pipeline" approach:
Vector removal
Assembly of identical and/or overlapping fragments
Identify genes
Lookup on genome if fully sequenced organism
Or genome contigs for partially sequences organsims
BLAST search of GeneBank for similar genes
Lookup in specialized database of "predicted genes"
ie. ENSEMBL
Project specific analysis
differentials between sets
Phylogenetics
DATABASE!!What these projects all share is a need to keep track of a lot of data
Hundreds to thousands of sequences
Many fields of information about each one
Organism, library, plate ID for each clone
the sequence itself
cluster/contig membership
best BLAST hit (accession #, e-value, alignment)
genome position
Can't keep track just using folders and text files on your hard drive
Design the database to include all possible fields
(it’s a lot harder to add info later)
Computer tools for sequencingA wide variety of different software tools have been created to aid DNA sequencing projects.
Each genome project lab has built its own custom software
UNIX
Based on a particular workflow design
PHRED, PHRAP, and Consed
Many packages for the individual investigator - included in most “comprehensive” molecular biology products: MacVector, LaserGene, DNA*, etc.
I will focus on the assembly tools in GCG, Consed and the dedicated sequence assembly program Sequencher
The GCG Fragment Assembly System
GCG has a complete set of programs that allow data entry, and assembly of overlapping nucleotide sequence fragments into one contig
SEQED: a single sequence editor
GELSTART: creates fragment assembly projects
GELENTER: adds sequences (reads) to an assembly project, input of new sequences from keyboard, digitizer, or import of existing text files
GELMERGE: assembles individual sequences into contigs, can automatically remove vector sequences
GELASSEMBLE: multiple sequence editor for viewing and editing contigs, allows manual alignment of fragments insertion/deletion of gaps and changing of individual bases
GELDISASSEMBLE: breaks up contigs into individual sequences within a project
GELVIEW: displays contigs as a schematic display of overlapping fragments
SeqLab has a Chromatogram viewer
Other Chromatogram Viewers
Applied Biosystems has a free viewer/editor program for sequence chromatograms
It is called EditView and it is a Macintosh only program (does not work in System 9.1 and newer)
http://cancer-seqbase.uchicago.edu/documents/EditView.hqx
There are a couple of viewers for Windows machines
ABIView is free from David H. Klatte
http:/http://www.bbioo.com/bioinformatics.weizmann.ac.il/software/abiview/abiinfo.html
Chromas is $50 shareware from Conor McCarthy, Technelysium Pty Ltd in Australia
http://www.technelysium.com.au/chromas.html
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