1. 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

2. A very short history of DNA sequencing

3. 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

4. Milestones First Isolation of DNA : 1867 (Freidrich Meisher)
Composition of nucleic acids; tetranucleotide theory : (Phoebus Levine)
G=C and A=T however, the G/C and A/T content of different organisms vary : 1950 (Edwin Chargaff)
G/C content measured by annealing : 1968 (Mandel and Marmur)
Maxam-Gilbert and Sanger Sequencing : 1977
Next-Generation Sequencing : 2005

5. 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)

6. Next-Generation Sequencing
Liu et al. Journal of Biomedicine and Biotechnology Volume 2012 (2012), Article ID , 11 pages doi: /2012/251364

7. Changes in instrument capacity*
ER Mardis. Nature 470, (2011) doi: /nature09796

8. Sequencing Cost Date Cost per Mb Cost per Genome Sep-01 $5,292.39
$95,263,072
Sep-02
$3,413.80
$61,448,422
Oct-03
$2,230.98
$40,157,554
Oct-04
$1,028.85
$18,519,312
Oct-05
$766.73
$13,801,124
Oct-06
$581.92
$10,474,556
Oct-07
$397.09
$7,147,571
Oct-08
$3.81
$342,502
Oct-09
$0.78
$70,333
Oct-10
$0.32
$29,092
Oct-11
$0.09
$7,743
Oct-12
$0.07
$6,618
Jan-13
$0.06
$5,671
Source - NHGRI :

9. Central Dogma of Molecular Biology
James Watson version
RNA
Protein
DNA
So once we have the genomic DNA sequence of a species we have all of the information there is?
Really?

10. No, not really.

12. Illumina HiSeq and MiSeq
Massively parallel
HiSeq : 150 or 180 million reads per lane
MiSeq : 15 million reads per run
Intermediate Read Length
HiSeq : 100 nt or 150 nt
MiSeq : 250 nt
High total output per run
HiSeq : 90 GB or 288 GB
MiSeq : 8 GB

13. Sequencing Types
Single Read
Paired-end read
Mate-pair read

14. Library Types
Many different library preps : DNA, mate-pair, mRNA, miRNA, ChIP
Fragmentation
DNA : 300 – 500 nt
RNA : 150 – 200 nt
Attachment of appropriate adapters
Complex : flow cell binding, F & R sequencing, BC
Custom : Avoid if possible
Removal of dimers/small inserts
Amplification (or not)

15. Applications de Novo sequencing (genomes, transcriptomes)
Resequencing (genomes, exomes, custom sequence capture)
RNA-seq (mRNA, miRNA, degradome)
Chip-Seq
Methyl-seq
RIP-seq
Amplicon

16. de Novo Experimental Design
Estimate of genome size
Coverage (30 x – 100 x)
Sequencing Type (paired-end or mate-pair)
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
Replicates

17. Resequencing : Sequence Capture

18. RNA-seq Experimental Design
Estimate of transcriptome size (1-5% of genome ?)
Coverage (30 x ?)
mRNA or rRNA depleted RNA
Relative abundance of transcripts you are interested in
Sequencing Type (single read or paired-end)
Simple transcriptome vs. complex transcriptome
Splice variants
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
Replicates : Yes!!!!
Biological not technical

19. ChIP-Seq

21. RIP-seq
Source :

22. Methyl-seq
20 different types of base modifications in DNA are known and there are perhaps 200 modifications of RNA

23. Experimental Space: Next-Gen Platform
PacBio : x 106 reads/sample, 1000 – 3000 nt
Whole transcript
Roche 454 FLX+ : x 106 reads/sample, 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 : x 106 reads per sample, 50 – 150 nt
Counting Applications : RNA-seq, ChIP-seq, RIP-seq, Methyl-seq
Large genome de novo and resequencing

24. Experimental Space: The Relevancy of “Classic” Techniques
Differential Gene Expression
Northern blotting (1977) : 1 Probe – 20 samples
Dot Blots (1987) : 100s of probes – 1 sample
RT-PCR (1992) : 100s of probes – samples
Microarrays (1995 ) : 100,000s of probes – 1 sample
Next-gen sequencing (2005) : x 106 reads – 1 sample

25. The Future More Reads Longer Reads Faster Sequencing
Cheaper Sequencing
New Applications