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Next-Generation Sequencing Technologies Overview

Explore the latest sequencing technologies including Molecular Inversion Probes, Illumina Genotype Arrays, and Nanopore Sequencing. Learn about Polony Sequencing, Solexa, and Pyrosequencing. Understand genome evolution through macro events.

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Next-Generation Sequencing Technologies Overview

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  1. Some new sequencing technologies

  2. Molecular Inversion Probes

  3. Illumina Genotype Arrays

  4. Single Molecule Array for Genotyping—Solexa

  5. Nanopore Sequencing http://www.mcb.harvard.edu/branton/index.htm

  6. Pyrosequencing on a chip • Mostafa Ronaghi, Stanford Genome Technologies Center • 454 Life Sciences

  7. Polony Sequencing

  8. Technologies available today • Illumina • 550,000 SNP array: $300-500 in bulk • 454 • 200 bp reads, 100 Mbp total sequence in 1 run, $8K • 500bp reads in much higher throughput coming soon • Solexa • 1Gbp of sequence coming in paired 35 bp reads • 1 day, approx $10K / run

  9. Short read sequencing protocol • Random, high-coverage clone library (CovG = 7 – 10x) • Low-coverage of clone by reads (CovR = 1 – 2x)

  10. Short read sequencing protocol

  11. Ordering clones into clone contigs

  12. Contig assembly

  13. Contig assembly

  14. Assembly quality Read length = 200 bp, Error rate = 1%, Net coverage = 20.0x

  15. Multiple Sequence Alignment

  16. Evolution at the DNA level Deletion Mutation …ACGGTGCAGTTACCA… SEQUENCE EDITS …AC----CAGTCCACCA… REARRANGEMENTS Inversion Translocation Duplication

  17. Evolutionary Rates next generation OK OK OK X X Still OK?

  18. Genome Evolution – Macro Events • Inversions • Deletions • Duplications

  19. Synteny maps Comparison of human and mouse

  20. Synteny maps

  21. Orthology, Paralogy, Inparalogs, Outparalogs

  22. Synteny maps

  23. Synteny maps

  24. Building synteny maps Recommended local aligners • BLASTZ • Most accurate, especially for genes • Chains local alignments • WU-BLAST • Good tradeoff of efficiency/sensitivity • Best command-line options • BLAT • Fast, less sensitive • Good for • comparing very similar sequences • finding rough homology map

  25. Index-based local alignment …… Dictionary: All words of length k (~10) Alignment initiated between words of alignment score  T (typically T = k) Alignment: Ungapped extensions until score below statistical threshold Output: All local alignments with score > statistical threshold query …… scan DB query Question: Using an idea from overlap detection, better way to find all local alignments between two genomes?

  26. Local Alignments

  27. After chaining

  28. Chaining local alignments • Find local alignments • Chain -O(NlogN) L.I.S. • Restricted DP

  29. Progressive Alignment x • When evolutionary tree is known: • Align closest first, in the order of the tree • In each step, align two sequences x, y, or profiles px, py, to generate a new alignment with associated profile presult Weighted version: • Tree edges have weights, proportional to the divergence in that edge • New profile is a weighted average of two old profiles y Example Profile: (A, C, G, T, -) px = (0.8, 0.2, 0, 0, 0) py = (0.6, 0, 0, 0, 0.4) s(px, py) = 0.8*0.6*s(A, A) + 0.2*0.6*s(C, A) + 0.8*0.4*s(A, -) + 0.2*0.4*s(C, -) Result:pxy= (0.7, 0.1, 0, 0, 0.2) s(px, -) = 0.8*1.0*s(A, -) + 0.2*1.0*s(C, -) Result:px-= (0.4, 0.1, 0, 0, 0.5) z w

  30. Threaded Blockset Aligner HMR – CD Restricted Area Profile Alignment Human–Cow

  31. Reconstructing the Ancestral Mammalian Genome Human: C C Baboon: C G Dog: G C or G Cat: C

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