Canadian Bioinformatics Workshops

Transcription

1 Canadian Bioinformatics Workshops www.

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3 Module 1

4 Overview next-gen or next-next-gen : why are we here? What kinds of sequencing are we doing? How does DNA sequencing works? Trying to stay away from vender-specific challenges, but can we really? Where next?

5 History of DNA Sequencing Adapted from Eric Green, NIH; Adapted from Messing & Llaca, PNAS (1998) Efficiency (bp/person/year) Miescher: Discovers DNA Avery: Proposes DNA as Genetic Material Watson & Crick: Double Helix Structure of DNA Holley: Sequences Yeast trna Ala ,500 15,000 25,000 50, , Wu: Sequences Cohesive End DNA Sanger: Dideoxy Chain Termination Gilbert: Chemical Degradation Messing: M13 Cloning Hood et al.: Partial Automation Cycle Sequencing Improved Sequencing Enzymes Improved Fluorescent Detection Schemes 50,000, ,000,000, Next Generation Sequencing Improved enzymes and chemistry New image processing

6 Why are we sequencing? Before Next-generation: Reductionist perspective on life DNA, RNA, (proteins), (populations), sampling, averages, consensus Problems: sampling, averages, consensus. After Next-generation: We are still reductionist, but better Genome sequence and structure Less cloning/pcr Single molecules (for some)

7 Basics of the old technology Clone the DNA. Generate a ladder of labeled (colored) molecules that are different by 1 nucleotide. Separate mixture on some matrix. Detect fluorochrome by laser. Interpret peaks as string of DNA. Strings are 500 to 1,000 letters long 1 machine generates 57,000 nucleotides/run Assemble all strings into a whole.

8 Whole Genome Sanger (old-gen) Sequencing Human (early drafts), model organisms, bacteria, viruses and mitochondria (chloroplast), low coverage Now-Gen Sequencing New human (!), individual genome, 1,000 normal, 25,000 cancer matched control pairs, rare-samples RNA cdna clones, ESTs, Full Length Insert cdnas, other RNAs RNA-Seq: Digitization of transcriptome, alternative splicing events, mirna Communities Environmental sampling, 16S RNA populations, ocean sampling, Human microbiome, deep environmental sequencing, Bar-Seq Other Epigenome, rearrangements, ChIP-Seq

9 Differences between the various platforms: Nanotechnology used. Resolution of the image analysis. Chemistry and enzymology. Signal to noise detection in the software Software/images/file size/pipeline Cost $$$

10 Adapted from Richard Wilson, School of Medicine, Washington University, Sequencing the Cancer Genome Next Generation DNA Sequencing Technologies Human Genome 6GB == 6000 MB Req d Coverage Illumina bp/read X75 reads/run , , bp/run 57, GB 15 GB # runs req d 625, runs/day Machine days/human genome 312,500 (856 years) Cost/run $48 $6,800 $9,300 Total cost $15,000,000 $979,200 $111,600

11 Next-gen sequencers From John McPherson, OICR 100 Gb 10 Gb AB/SOLiDv3, Illumina/GAII short-read sequencers (10+Gb in bp reads, >100M reads, 4-8 days) bases per machine run 1 Gb 100 Mb 10 Mb 1 Mb 454 GS FLX pyrosequencer ( Mb in bp reads, 0.5-1M reads, 5-10 hours) ABI capillary sequencer ( Mb in bp reads, 96 reads, 1-3 hours) 10 bp 100 bp 1,000 bp read length

12 From John McPherson, OICR 2009/10 Promises? 100 Gb 10 Gb AB SOLiDv3 120Gb, 100 bp reads Illumina GAII 90Gb, 175bp reads bases per machine run 1 Gb 100 Mb 10 Mb 1 Mb 454 GS FLX Titanium Gb, bp reads ABI capillary sequencer ( Mb, bp reads 10 bp 100 bp 1,000 bp read length

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14 Adapted from Richard Wilson, School of Medicine, Washington University, Sequencing the Cancer Genome Solexa-based Whole Genome Sequencing

15 Illumina (Solexa)

16 Illumina (Solexa)

17 Illumina (Solexa)

18 From Debbie Nickerson, Department of Genome Sciences, University of Washington,

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20 AB SOLiD: file management

21 SOLiD color space

22 SOLiD color space

23 SOLiD color space

24 SOLiD color space

25 SOLiD color space

26 SOLiD color space

27 AB SOLiD

28 SOLiD color space

29 SOLiD color space

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31 SOLiD color space

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33 SOLiD color space

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36 >443_1087_001_F3 T >443_1087_002_F3 T >443_1087_003_F3 T >443_1087_004_F3 T >443_1088_005_F3 T >443_1088_006_F3 T >443_1088_007_F3 T Sample AB data Lab Get sequence assignment from instructor Work with people at your table. Use info from lecture notes (Panel E) BLAST sequence at NCBI What is it? >443_1088_008_F3 T >443_1088_009_F3 T >443_1088_010_F3 T

37 Module 1 lab

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39 Roche / 454 : GS FLX Also known as pyrosequencing million bp/run 10 hr run bp/read & > 1 M reads

40 Roche / 454 : GS FLX Made for de novo sequencing. Too expensive for resequencing. For example, this platform will be used a lot by laboratories doing new bacterial genomes. Baylor Genome Center involved in Sea Urchin, Bee, Platypus genomes: They have a number of 454.

41 Roche / 454 : GS FLX

42 Roche / 454 : GS FLX

43 Roche / 454 : GS FLX

44 It s more complicated! Get files with quality scores Get files with miss-matches Need to align them to a reference genome Multiple tools do this today and there will be more later. What do you do? Do it all!

45 Pacific Biosystems (PacBio) July 2008

46 Pacific Biosystems (PacBio)

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49 Things to keep in mind All people are learning, if you don t know, ask, and they probably won t know either, and you can figure it out together! The technology is changing This workshop next year will be totally different! We can only do so much in two days you will need to find things, find people who can help you, and you will need to teach your friends!

50 Other factors Changing technology New and disappearing companies? Changing price structure Cost of machine Cost of operation (reagents/people) Service from the company 1 machine vs (2 or 3 machines) vs 40 machines. Changing software and processing

51 OICR Informatics: servers, CPU, Storage, and Backups Storage 125 X Web Dev SVN 14 Sequencers cluster 50 X local (150 GB) 10 X seq (9 TB) FC (25 TB) N-series SATA (25 TB) BlueArc SATA (1PB) SAS (40 TB) 12 X MS- Windows 200 X 5 X 8 core 16 GB RAM 8 core 96 or 256 GB RAM 1640 cores 1259 TB Back Up Robot 800 GB/tape 12 Drives > 300 tape library

52 What have we learned? Sequencing technologies are changing fast Allowing new biology to be performed, new questions to be asked Understand the difference between some of the technologies You can work in color space.

53 What next?

54 Day 1

55 URLs