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

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

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

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

Sanger Sequencing

DNA is fragmentedCloned to a vector

Plasmid, BACLinkage

Cyclic sequencingSeparation byelectrophoresisRead fluorescent tags