MICR 4280 Lecture Notes - Lecture 10: Massively Parallel (Computing), High Fidelity, Consensus Sequence
19 Mar 2019
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Wednesday january 30 t h lecture 10. 4 basic steps: sample prep, cluster generation, sequencing, and data analysis. Sample prep: add adaptors to end of dna: cluster generation: isothermally amplified, complimentary oligo, clonal amplification. Cluster generation using o29 dna polymerase: high fidelity and displaces bases isothermal (non-pcr) amplification. Pacific bioscience/p acbio libra ry preperartion: at the ends of the template dna, ligate the single stranded adaptors which give you hairpin shape, they are single stranded, gives you a covalently closed circle (smrtbell) Smrt cell: chip containing 150,000 zero-mode waveguide (zmw) Smrtbell, polymerase anchors to the bottom of the zmw, since covalently closed circle, you can replicate all the way through the molecule and then start again. Once you hit the adaptor, you know you are entering the complementary strand. Circular dna allows polymerase to continuously move in circles. Pacbio -single-molecule real-time (smrt) sequencing via ligh t pulses:
