Transcript Faber: Sequence resources

Methods in genome sequencing
and SNP finding
Gabor Marth
BI 820
presented by Tony Faber
Sequences used in SNP analysis and
genomic sequencing
Expressed Sequence Tags (ESTs)
 Sequence-tagged site (STS) sequences
 Reduced Representation Libraries (RRL)
 Whole-genome shotgun libraries
 Genome Survey Sequence (GSS)
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Expressed Sequence Tags (ESTs)
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Relatively short (200-400bp) of partial cDNA sequences
Many are single-pass reads from tissue specific cDNA
libraries
HGP aligned to human reference sequence EST quality
(SEQREF)
Coding and UTRs make up ESTs (can be
multiple exons)
Identifying putative full-ORF
cDNA clones
5’ ESTs
Matches Refseq
No
Yes
Read
page
2
5’ end aligns at start
no
Yes
Protein Comp
Matches
Aminoterminus
HKScan
GenomeScan Comparision
Matches 5”
end of
predicted
gene
Select for complete sequencing
Sequenced Tagged Sites (STSs)
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First used- advantages include PCR primers readily
available, recovered BACs/YACs during HGP PCR much
cheaper than BAC/YAC sequencing
Represent the superposition (i.e. can also be double-pass
reads)
Fingerprint clone contigs bound to specific STSs
Whole-genome shotgun
• Random clones from the genomes of many individuals
• Requires several-fold coverage of the genome (e.g.
sequencing, SNP discovery)
Genome Survey Sequence (GSS)
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To survey a new genome, or get a general idea of genomic
make-up of organism
Similar to ESTs, except the DNA is genomic in origin (not
mRNA)
Also single pass reads
From cosmid/BAC/YAC ends, exon trapped genomic
sequences, and Alu PCR sequences
Splicing events
Reduced Representation sequences
(RRS)
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Heavy cloning in certain regions
Contain STSs, many corresponding to genes or ESTs
One clone per MB on every chromosome, excellent coverage
Reproducibly prepared subsets of the genome from several
individuals, each containing a manageable number of loci
Thus allowing Re-sampling
Greater flexibility and efficiency
Problems- creating reduced representations, finding ortholouges
matches, accuracy
Origin of replication
Binding to particular protein
Restriction fragments in a certain range (size selected restriction
fragments)
Chromosome 12
SNP context
• Most popular method for obtaining SNP’s
•EST alignment
•Major sources of genomic SNPs include sequences for
restricted genome representation libraries, random
shotgun reads aligned to genome sequence, BAC/YAC
overlaps