DNA sequencing using polymerase substrate-binding kinetics (Nature Communications)

Intro

illumina sequences

• build clusters. deblock of 3′ site, adding of base. remove base. Deblock site. add base

ion-torrent –

• natural nucleotides – faster, no extra chemistry, cheaper, longer read lengths, do require oil emulsions. faster run time. (10-20x more expensive)

New method

• measure signal from fluorescent polymerase bound, use difference in kinetics of correct vs incorrect base to measure sequence
• up to 3 fold difference in fluorescence at high salt (in bulk)
• up to 5 fold difference in fluorescence observed on glass
• homopolymer calling: kinetic difference and total fluorescence combined help distinguish homopolymers
  • better for 1 vs 2 vs 3 than 3 vs 4 (went fast)
• 44 rounds of flowing bases, sequence bases
• sequence phage genome (clip reads at 20bp). 95% accuracy in non-homopolymeric regions, 90% accuracy in homopolymeric regions.

for improvement

• better engineered enzyme
• improve base calling algorithm (correct for local sequence context)
• calibration sequences – normalize for sequence size
• surface modifications (does labeled polymerase accumulate stuck to the surface?)
• better fluidic mixing and flow?