Bio-5488_next_gen_seq_2010_2.pptVIP

Next Generation Sequencing Technologies Rob Mitra 5488 Lecture 1/24/10 Lecture Outline Odds and Ends from Next Generation Sequencing Lecture 1 Third Generation Sequencing The Poisson Distribution and Fold Coverage Aligning Reads to a Reference - A Practical Introduction Read Length is Not As Important For Resequencing Sequencing a complete cancer genome What did we learn? Mutation spectrum carries a signature of damage by UV light. Transcription coupled repair is a important DNA repair mechanism. Plausible candidate genes. What will we learn? What will we learn part 2? In non-small cell lung cancer, the tyrosine kinase domain of EGFR is mutated in some patients (often non-smokers) This mutation activates an anti-apoptotic pathway The tyrosine kinase inhibitor Gefitinib is effective in patients with this mutation (60% show no growth or remission after 1 year versus 7% on chemotherapy) Very few side effects! Personalized Therapy Pacific Biosciences: A Third Generation Sequencing Technology Real Time Sequencing How did they do? 150 bp circular template ~93% raw accuracy 15x coverage 99.3% accuracy Still early days Where are they going? Commercial specifications: 80,000 zero mode waveguides 10-15 minute runs Throughput ~140 MB per hour* (currently Illumina is at ~100 MB per hour,expect 625 MB per hour this summer) Methyl C? Dark reads? Phi29 so long read lengths possible (3kb now, up to 70kb later?) Ease of sample prep Camera costs Math Aside: Sequencing coverage calculations Let’s say you need a base to be sequenced 5x for an accurate base call If you sequence at 10x coverage how much of the genome will be sequenced at least 5 times? Poisson Distribution Example Average coverage = 5x Probability of a given base being sequenced exactly 10 times is: 510e-5/10! = 0.018 or about 2% of bases will have 10x coverage. Math Aside: Sequencing coverage calculations If you sequence at 10x coverage how much of the genome will be sequenced at least 5 times?