1 Next Generation Sequencing Itai Sharon November 11th, 2009 Introduction to Bioinformatics.

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Presentation transcript:

1 Next Generation Sequencing Itai Sharon November 11th, 2009 Introduction to Bioinformatics

2 2010: 5K$, a few days? 2009: Illumina, Helicos 40-50K$ Sequencing the Human Genome Year Log 10 (price) : 100$, <24 hrs? 2008: ABI SOLiD 60K$, 2 weeks 2007: 454 1M$, 3 months 2001: Celera 100M$, 3 years 2001: Human Genome Project 2.7G$, 11 years

3 In this Talk: Sequencing 1.0: Sanger Assembly Next generation sequencing (NGS) NGS applications Future directions

Genome Sequencing Goal  figuring the order of nucleotides across a genome Problem  Current DNA sequencing methods can handle only short stretches of DNA at once (<1-2Kbp) Solution  Sequence and then use computers to assemble the small pieces 4

Genome Sequencing 5 5 ACGTGGTAA CGTATACAC TAGGCCATA GTAATGGCG CACCCTTAG TGGCGTATA CATA… ACGTGGTAATGGCGTATACACCCTTAGGCCATA Short fragments of DNA AC..GC TT..TC CG..CA AC..GC TG..GTTC..CC GA..GC TG..AC CT..TG GT..GC AC..GC AT..AT TT..CC AA..GC Short DNA sequences ACGTGACCGGTACTGGTAACGTACA CCTACGTGACCGGTACTGGTAACGT ACGCCTACGTGACCGGTACTGGTAA CGTATACACGTGACCGGTACTGGTA ACGTACACCTACGTGACCGGTACTG GTAACGTACGCCTACGTGACCGGTA CTGGTAACGTATACCTCT... Sequenced genome Genome

Sanger Sequencing Mix DNA with dNTPs and ddNTPs Amplify Run in Gel – Fragments migrate distance that is proportional to their size 6

Sanger Sequencing 7

Advantages  Long reads (~900bps)  Suitable for small projects Disadvantages  Low throughput  Expensive 8

Assembly 9 9 Cut DNA to larger pieces (2Kbp, 15Kbp) and sequence both ends of each piece (Fleischmann et al., 1994) contig 1 contig 2 15Kbp mates 2Kbp mates ~(length―1,000) ~500 bp resolving repeats Better assembly of contigs, gap lengths estimation

 many pieces to assemble High coverage: Assembly: How Much DNA? 10 Low coverage: A few pieces to assemble a few contigs, a few gaps many contigs, many gaps  Input Output Lander and Waterman, 1988

Sanger Sequencing : lambda virus DNA stretches up to 30-40Kbp (Sanger et al.) 1994: H. Influenzae 1.8 Mbp (Fleischmann et al.) 2001: H. Sapiens, D. Melanogaster 3 Gbp (Venter et al.) 2007: Global Ocean Sampling Expedition ~3,000 organisms, 7Gbp (Venter et al.)

12 Next Generation Sequencing: Why Now? Motivation: HGP and its derivatives, personalized medicine Short reads applications: (re-)sequencing, other methods (e.g. gene expression) Advancements in technology

13 High Parallelism is Achieved in Polony Sequencing PolonySanger

14 Generation of Polony array: DNA Beads (454, SOLiD) DNA Beads are generated using Emulsion PCR

15 Generation of Polony array: DNA Beads (454, SOLiD) DNA Beads are placed in wells

16 Generation of Polony array: Bridge-PCR (Solexa) DNA fragments are attached to array and used as PCR templates

17 Sequencing: Pyrosequencing (454) Complementary strand elongation: DNA Polymerase

18 Sequencing: Fluorescently labeled Nucleotides (Solexa) Complementary strand elongation: DNA Polymerase

19 Sequencing: Fluorescently Labeled Nucleotides (ABI SOLiD) Complementary strand elongation: DNA Ligase

20 Sequencing: Fluorescently Labeled Nucleotides (ABI SOLiD) 5 reading frames, each position is read twice

21 Single Molecule Sequencing: HeliScope Direct sequencing of DNA molecules: no amplification stage DNA fragments are attached to array Potential benefits: higher throughput, less errors

22 Technology Summary Read lengthSequencing Technology Throughput (per run) Cost (1mbp)* Sanger~800bpSanger400kbp500$ 454~400bpPolony500Mbp60$ Solexa75bpPolony20Gbp2$ SOLiD75bpPolony60Gbp2$ Helicos30-35bpSingle molecule 25Gbp1$ *Source: Shendure & Ji, Nat Biotech, 2008

23 What, When and Why Sanger: Small projects (less than 1Mbp) 454: De-novo sequencing, metagenomics Solexa, SOLiD, Heliscope: – Gene expression, protein-DNA interactions – Resequencing

24 Applications

25 Applications

26 Where Do We Go from Here? Higher throughput, longer reads (Pacific BioSciences) Computational bottleneck Shift to sequencing-based technologies Will it help to cure cancer?