Next Generation Sequencing

Transcription

1 Next Generation Sequencing Molecular Methods Sylvain Forêt March

2 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

3 The Genomic Age Recent landmarks in genomics 1995 First bacterial genome (1.8 Mb) 1996 First eukaryotic genome (12 Mb) 1998 First animal genome (100 Mb) 2000 First human genome (3 Gb)

4 The Post-Genomic Age Two big questions How can we continue sequencing ever faster? What can be done with all these sequences? The Archon X Prize To win the prize purse, the registered group must build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced for no more than $10,000 per genome. Other challenges and projects The $1,000 human genome The 1,000 genomes project (NHGRI, BGI,... )

5 Course Outline Next generation (massively parallel) sequencing Molecular methods (course 1) Applications (course 2, course 3)

6 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

7 Sanger Method Template Synthesis Primer DNA polymerase T G G T A T C C G T A C C G A Electrophoresis T T T T A A C A C G Chromatogram Quality score

8 Sanger Method Summary Main characteristics Sequencing by synthesis Dye terminator method Input Material: any DNA in sufficient quantity PCR products Molecular clones...

9 Error and Quality Sources of error Material (contamination, polymorphism, etc) DNA polymerase Signal (more prevalent at the end of the sequences)

10 Error and Quality Quality scores Each base sequenced is assigned a quality score Q By definition: Q = 10 log 10 (probability of error) Thus: probability of error = 10 Q 10 Quality score Position

11 Error and Quality Consequences Only relatively small sequences can be sequenced Long sequences must be sequenced in several steps For accuracy, each based should be covered more than once Histogram of sizes, mean = N50 = 762 Density Size

12 Shotgun sequencing DNA extraction DNA fragmentation Cloning into vectors Vector Primers Grow vector in bacteria Insert Extract and sequence vectors Map or assemble sequences Mate pairs

13 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

14 Illumina: Sample Preparation Biological Sample DNA extraction RNA fragmentation, size selection fragmentation, size selection reverse transcription (random primers)

15 Illumina: Sequencing (1) Source:

16 Illumina: Sequencing (2) Source:

17 Illumina: Sequencing (3) Source:

18 Illumina: Sequencing (4) Source:

19 Illumina: Mate Pairs

20 Illumina: Multiplexing Multiplexing Flow cell: 8 lanes Each lane: up to 96 samples Source:

21 Illumina Summary Main characteristics Sequencing by synthesis Reversible terminator method Current size: 100bp

22 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

23 Pyrosequencing Chemistry + A Template + Nucleotides Extended template + PPi (pyrophosphate) PPi + ADP phosphosulfate + sulfurylate ATP ATP + Luciferin + Luciferase Oxyluciferin

24 454 Pyrosequencing From: Margulis et al, Nature 2005

25 454: Poly-A Tails AAAAAAAAAAA TTTTTTTTTTT RE AAAAAAAAAAA TTTTTTTTTTT RE AAAAAAAAAAA TTGTTTCTTTT

26 454: Mate Pairs Internal adapter Insert (3kb 20kb) Internal adapter Circularize bp Cut bp Add sequencing adapters Sequence

27 454: Multiplexing Multiplexing Each plate: 1, 2, 4, 8 or 16 regions separated by gaskets Each region: up to 12 samples Adapter Template Adapter + MID Template (Multiplex Identifier) Source:

28 454 Summary Main characteristics Sequencing by synthesis Pyrosequencing method Current size: 400bp

29 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

30 SOLiD: Sequencing (1) Source:

31 SOLiD: Sequencing (2) Source:

32 SOLiD: Sequencing (3) Source:

33 SOLiD: Sequencing (4) Source:

34 SOLiD: Sequencing (5) Source:

35 SOLiD: Mate Pairs Internal adapter Insert (600bp 10kb) Internal adapter Circularize Cut Add sequencing adapters Sequence

36 SOLiD: Multiplexing Multiplexing Each run: 2 slides Each slides: 1, 2, 4, 8 regions Each region: up to 16 samples Source:

37 SOLiD Summary Main characteristics Sequencing by ligation Current size: 50bp

38 1 Introduction 2 Sanger 3 Illumina SOLiD 6 Summary

39 Summary Numbers, as of March Illumina SOLiD (Titanium) (Genome Analyser II x ) (SOLiD 3) Mean read size 400bp 100bp 50 bp Reads per run Run time 10 hours 4 days 1 week Insert size 3kb 20kb 200bp 5kb 600bp 10kb

40 Summary Conclusions Fast moving field Other players: Helicos, Pacific Biosciences, Nano Pores,... A $1,000 human genome seems possible within a few years Many applications Genome (re)sequencing Transcriptome sequencing ChIP-seq Metagenomics...