Stratton Nature 45: 719, 2009Evolution of DNA sequencing technologies - 1980 to present day DNA SEQUENCING & ASSEMBLY

The X Prize Foundation of Playa Vista, California, is offering a $10-million prize to the first team to accurately sequence the genomes of 100 people aged 100 or older, for $1,000 or less apiece and within 30 days [beginning September 5, 2013]. see Nature 487:417, July 26, 2012$$$ Motivation to spur DNA sequencing technologies, boost accuracy and drive down costs with an accuracy of

but it stops when dideoxynucleotideis incorporated4 parallel sets of reactions: ddATP + 4 dNTPsddCTP + 4 dNTPs etc.Fig. 4.2Sanger chain termination method (Fred Sanger, 1977)- enzymatic synthesis of DNA strand complementary to template of interestNobel Prizes: Sanger 1958 (protein structure)1980 (DNA sequencing)

Fig. 4.2Ratio of ddATP:dATP importantto get appropriate size range of products- set of products each terminating with ddA- their sizes reflect positions of T in template DNA

Products (each differing in length by 1 nt) resolved on denaturing polyacrylamide gels...Automated sequencing profileAutoradiographFig. 4.1Fig. 4.3or by capillary electrophoresis

Fig. 4.5PRIMERS FOR SEQUENCING1. Universal - forward & reverse2. Custom-designed internal use new sequence info to design primer to sequence next stretchIf insert is too long to completely sequence using universal primers, can use this strategy to close a sequencing gap

or can find another clone in library that has overlap, and sequence it using universal primersFig. 3.35

if particular region of genome is not represented in clone library can use a different vector to prepare a second clone library then use probes (eg. oligomers) mapping to ends of contigs from first library to screen second library(maybe region was unstable in first vector)What if there is a physical gap?Fig. 4.17

Fig. 4.11Which contigs are adjacent? or by PCRYou have 9 contigs & design oligomers mapping close to their ends (#1-18)Example of closing a physical gapscreening by hybridization 8 7 1 2

What if physical gap is very short?- then sequence the PCR product directly- could use oligomers mapping to ends of contigs in PCRreactions with uncloned DNA template3 55 3 < 10 kb or so- this slide also illustrates a method for finding overlapping clones

Fig. 4.12- repeat to walk along genomeASSEMBLING INFO FROM CLONES INTO CONTIGS1. CHROMOSOME WALKING by hybridization- sequence from one clone is used as probe to screenlibrary of clones to find overlapping one

But what if probe contains repeated sequences?Problem avoided if use short unique-sequence probe(eg oligomer) mapping close to end of clone- so hybridizes to multiple clones or if pre-hybridize with repeat sequenceFig. 3.34

2. CHROMOSOME WALKING by PCRFig. 4.13- reactions can be carried out as pools for more rapid screening- design primer pairs based on sequence at end of clone- use other clones in library for template DNA- will get PCR amplicon for any new clones with that sequence(combinatorial screening) Fig. 4.14

Fig. 4.15A3. CLONE FINGERPRINTINGRestriction profile fingerprint To identify overlapping clones: by finding features that they shareor clones having STS in common (Fig.4.15D)

Fig. 4.10Haemophilus genome project 1995 (1.8 Mbp)1. DNA sonicated, fragments (1.6 2 kb) cloned in plasmid vectors2. Shotgun sequencing of insert ends~ 20,000 clones analyzed, 11 Mbp of sequence, scaffolds with sequencing gaps & physical gaps4. Screened for overlapping clones reduced to 42 contigs3. Assembled into 140 contigs5. Assumed gaps represented genome regions unstable in plasmid vector - switched to lambda vector6. Probed l library with oligomers from contig ends or used PCR with primer pairs from contig ends

Cost per Megabase of DNA Sequence (or Why biologists panic about computing) Next generation sequencing technologiesNational Human Genome Research Institute - major challenge to correctly assemble the massive amount of sequence data generatedand to interpret it !

Genome Res 11:3, 2001- one dNTP is added at a time + enzyme (apyrase) that degradesdNTP if not incorporated into new strand, then next dNTP added - incorporation detected by chemiluminescence of pyrophosphate (PPi)Fig. 4.91. PyrosequencingCwww.youtube.com/watch?v=kYAGFrbGl6E&feature=related

Medini Nat Rev Microbiol. 6:419, 2008- DNA sheared, adaptors ligated, attached to bead & PCR amplified- beads captured in wells & pyrosequencing carried out in parallel on each DNA fragmentEnzymes on beads and primerSample preparationPyrosequencingPCRPolymerasePPiLightGenomic DNA- average read of ~ 700 (?) bpMassively-parallel pyrosequencing (on beads or chips)454 technology ... but up to 1.6 million reactions can be carried out in parallel on a 6.4 cm2 slideexpect ~ 500 million nucleotides of sequence data per 10 hour run (July 2010)

2. Illumina sequencing (parallel microchip)- average read of ~ 40-100 bp (short-read) - add adaptors to sheared DNA, attach to chip, then PCR bridge amplification- denature clusters of ~ 1000 copies of DNA molecules & sequential sequencing using four fluorophore-labelled ntsSOLEXA technologyMedini Nat Rev Microbiol. 6:419, 2008www.youtube.com/watch?v=HtuUFUnYB9Y&feature=relatedHiSeq 2000 HiSeq 1000(Illumina website Sept. 2012)

Output (2 100 bp)600 Gb300 GbRun Time (2 100 bp) ~11 days~8.5 daysPaired-end Reads6 Billion3 BillionSingle Reads3 Billion1.5 BillionMaximum Read Length**2 100 bp2 100 bpBases Above Q30***> 85% (2 x 50 bp) > 80% (2 x 100 bp)

3. Single molecule real-time sequencing (Helicos, Pacific Biosciences)Metzker Nature Reviews Genetics 11:31, 2010- continuous monitoring of nt incorporation (rather than termination as in Sanger method) and no amplification- formation of phosphodiester bond releases fluorophore- nanoscale wells on chip so ~ one DNA polymerase molecule per well(Helicos website Sept. 2012)- read length 25 to 55 bases, 21-35 Gigabases per run

Chin et al. New Eng J Med 364:33, 2011Press release, Dec 9,2010: PacBio & Harvard Use Fast Gene Sequencer to Crack DNA Code of Haitian Cholera StrainH1 and H2 strains were sequenced in < 24 hr with enough reads to cover the genomes 60 and 32 times, respectively.