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The Evolution of SequencingSanger sequencing
GelsCycle sequencingFluorescenceCapillary electrophoresis
Sequencing, the “Next Generation”“Sequencing by synthesis”
Pyrosequencing (Roche/454)Cluster sequencing (Illumina/Solexa)
Sequencing by ligation (ABI/SOLiD)Next “Next Generation”
4th Generation?Single molecule sequencing
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Macro versus Micro Reads
Illumina/SolexaGeneticAnalyzer
ReadLength35‐75bp<=>250‐450bp
BasePairsPerRun3‐10Gb<=>0.1‐0.5Gb
BasePairsPerDay1‐1.5Gb<=>0.2‐1.0Gb
NumberofSequences100M<=>1.2M
RunTime3‐7days<=>0.5days
ReagentCostperRun~$4K‐$12K<=>$6K
ErrorRateVaries,differentcharacteristics
AppliedBiosystemsSOLiD
Roche/454FLX
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Technology and InformaticsPR Space versus Science Space
Flow and phasingData quality and Error rate
Variation along sequenceQuality scores (Equivalence?)
Length distribution versus averageRaw versus recovered sequence
How much coverage with different methods?Tagging (barcodes) and multiplexing
Variation in coverage
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Next-Gen BasicsLibrary creation
Shearing, size selectionSize distribution
Specific primer sequences (adaptors) flank targetsequence
Allows amplificationOpportunity for extra “mutation”
Tagging (barcodes)Proportion of sequence wasted
Ligation or amplification (454)Paired ends
PrimerAOptionalBarcode
Key Libraryfragment PrimerB
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Sanger Sequencing
DNA is fragmentedCloned to a vector
Plasmid, BACLinkage
Cyclic sequencingSeparation byelectrophoresisRead fluorescent tags