The Past, Present, and Future of DNA Sequencing Craig A. Praul Co- Director Genomics Core Facility Huck Institutes of the Life Sciences Penn State University
A very short history of DNA sequencing
I started from the conviction that, if different DNA species exhibited different biological activities, there should also exist chemically demonstrable differences between deoxyribonucleic acids. Edwin Chargaff
Milestones •
First Isolation of DNA : 1867 (Freidrich Meisher)
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Composition of nucleic acids; tetranucleotide theory : 1909 - 1940 (Phoebus Levine)
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G=C and A=T however, the G/C and A/T content of different organisms vary : 1950 (Edwin Chargaff)
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G/C content measured by annealing : 1968 (Mandel and Marmur)
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Maxam-Gilbert and Sanger Sequencing : 1977
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Next-Generation Sequencing : 2005
Genomes Sequenced
• Virus – 3222 (Bacteriophage phi X 174, 5386 nt – 1977) • Bacteria – 2289 (Haemophilus influenza, 1.8 x 106 nt – 1995) • Eukarya – 168 (S. cerevisiae 1.2 x 107 nt – 1995; H. sapien, 3 x 109 nt -2001) • Archaea – 152 (Methanococcus jannaschi , 1.7 x 106 nt – 1996)
Next-Generation Sequencing
Liu et al. Journal of Biomedicine and Biotechnology Volume 2012 (2012), Article ID 251364, 11 pages doi:10.1155/2012/251364
Changes in instrument capacity*
ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796
Sequencing Cost Date Sep-01 Sep-02 Oct-03 Oct-04 Oct-05 Oct-06 Oct-07 Oct-08 Oct-09 Oct-10 Oct-11 Oct-12 Jan-13
Cost per Mb
Cost per Genome
$5,292.39 $3,413.80 $2,230.98 $1,028.85 $766.73 $581.92 $397.09 $3.81 $0.78 $0.32 $0.09 $0.07 $0.06
Source - NHGRI : http://www.genome.gov/sequencingcosts/
$95,263,072 $61,448,422 $40,157,554 $18,519,312 $13,801,124 $10,474,556 $7,147,571 $342,502 $70,333 $29,092 $7,743 $6,618 $5,671
Central Dogma of Molecular Biology James Watson version - 1965
DNA
RN Protein A So once we have the genomic DNA sequence of a species we have all of the information there is?
Really?
• No, not really.
Illumina HiSeq and MiSeq •
Massively parallel – HiSeq : 150 or 180 million reads per lane – MiSeq : 15 million reads per run
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Intermediate Read Length – HiSeq : 100 nt or 150 nt – MiSeq : 250 nt
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High total output per run – HiSeq : 90 GB or 288 GB – MiSeq : 8 GB
Sequencing Types
Single Read
Paired-end read
Mate-pair read
Library Types •
Many different library preps : DNA, mate-pair, mRNA, miRNA, ChIP
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Fragmentation – DNA : 300 – 500 nt – RNA : 150 – 200 nt
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Attachment of appropriate adapters – Complex : flow cell binding, F & R sequencing, BC – Custom : Avoid if possible
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Removal of dimers/small inserts
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Amplification (or not)
Applications •
de Novo sequencing (genomes, transcriptomes)
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Resequencing (genomes, exomes, custom sequence capture)
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RNA-seq (mRNA, miRNA, degradome)
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Chip-Seq
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Methyl-seq
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RIP-seq
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Amplicon
de Novo Experimental Design •
Estimate of genome size
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Coverage (30 x – 100 x)
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Sequencing Type (paired-end or mate-pair)
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Example 100 MB genome, 100 x 100 nt paired-end reads – (100 MB) x (30 x coverage) = 3 GB – 3 GB / (200 nt for each pair of paired-end reads) = 15 million read pairs
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Replicates
Resequencing : Sequence Capture
RNA-seq Experimental Design •
Estimate of transcriptome size (1-5% of genome ?)
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Coverage (30 x ?) – mRNA or rRNA depleted RNA – Relative abundance of transcripts you are interested in
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Sequencing Type (single read or paired-end) – Simple transcriptome vs. complex transcriptome – Splice variants
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Example 3 GB genome, 100 nt single reads – (3 GB genome) x ( 5% transcriptome ) = 120 MB Transcriptome – (120 MB transcriptome) x (30 x coverage) = 4.5 GB total sequence – 4.5 GB / (100 nt for each read) = 45 million read pairs
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Replicates : Yes!!!! – Biological not technical
ChIP-Seq
http://www.nature.com/nmeth/journal/v4/n8/images/nmeth0807-613-F1.gif
RIP-seq
Source : http://openi.nlm.nih.gov/imgs/rescaled512/3269675_ijms-13-00097f6.png
Methyl-seq
20 different types of base modifications in DNA are known and there are perhaps 200 modifications of RNA
Experimental Space: Next-Gen Platform • PacBio : 0.075 x 106 reads/sample, 1000 – 3000 nt – Whole transcript • Roche 454 FLX+ : 0.5 -1 x 106 reads/sample, 800 -1000 nt – Small – Medium Genome de novo sequencing – Long Amplicon – Transcriptome • PGM: 1-2 x 106 reads per sample, 400 nt – Small genome de novo – Medium Amplicon • MiSeq: 1-2 x 106 reads per sample, 50 – 250 nt – Small genome de Novo – Small Amplicon • HiSeq : 10-100 x 106 reads per sample, 50 – 150 nt – Counting Applications : RNA-seq, ChIP-seq, RIP-seq, Methyl-seq – Large genome de novo and resequencing
Experimental Space: The Relevancy of “Classic” Techniques Differential Gene Expression
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Northern blotting (1977) : 1 Probe – 20 samples
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Dot Blots (1987) : 100s of probes – 1 sample
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RT-PCR (1992) : 100s of probes – 10 -100 samples
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Microarrays (1995 ) : 100,000s of probes – 1 sample
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Next-gen sequencing (2005) : 10-100 x 106 reads – 1 sample
The Future
• More Reads • Longer Reads • Faster Sequencing • Cheaper Sequencing • New Applications