1. CS 6293 Advanced Topics: Current Bioinformatics
Next-generation sequencing - technology

2. Outline First generation sequencing
Next generation sequencing (current)
AKA:
Second generation sequencing
Massively parallel sequencing
Ultra high-throughput sequencing
Future generation sequencing
Analysis challenges

3. Sanger sequencing (1st generation)
DNA is fragmented
Cloned to a plasmid vector
Cyclic sequencing reaction
Separation by electrophoresis
Readout with fluorescent tags
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, (2008)

4. Cyclic-array methods (next-generation)
DNA is fragmented
Adaptors ligated to fragments
Several possible protocols yield array of PCR colonies.
Enyzmatic extension with fluorescently tagged nucleotides.
Cyclic readout by imaging the array.
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, (2008)

5. Available next-generation sequencing platforms
Illumina/Solexa
ABI SOLiD
Roche 454
Polonator
HeliScope … 5

6. Emulsion PCR
Fragments, with adaptors, are PCR amplified within a water drop in oil.
One primer is attached to the surface of a bead.
Used by 454, Polonator and SOLiD.
Rothberg and Leomon Nat Biotechnol. 2008
Shendure and Ji Nat Biotechnol. 2008

7. 454 Sequencing Stats: read lengths 200-300 bp
accuracy problem with homopolymers
400,000 reads per run
costs $60 per megabase
Rothberg and Leomon Nat Biotechnol. 2008

8. Bridge PCR DNA fragments are flanked with adaptors.
A flat surface coated with two types of primers, corresponding to the adaptors.
Amplification proceeds in cycles, with one end of each bridge tethered to the surface.
Used by illumina/Solexa.

10. All 4 labeled nucleotides
First Round
All 4 labeled nucleotides
Primers
Polymerase

11. 1. Take image of first cycle
2. Remove fluorophore
3. Remove block on 3’ terminus

13. Stats:
read lengths up to 36 bp
error rates 1-1.5%
several million “spots” per lane (8 lanes)
cost $2 per megabase

14. Conventional sequencing
Can sequence up to 1,000 bp, and per-base 'raw' accuracies as high as %. In the context of high-throughput shotgun genomic sequencing, Sanger sequencing costs on the order of $0.50 per kilobase.
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, (2008) 14

15. Sequence qualities
In most cases, the quality is poorest toward the ends, with a region of high quality in the middle
Uses of sequence qualities
‘Trimming’ of reads
Removal of low quality ends
Consensus calling in sequence assembly
Confidence metric for variant discovery
In general, newer approaches produce larger amounts of sequences that are shorter and of lower per-base quality
Next-generation sequencing has error rate around 1% or higher

16. Phred Quality Score p=error probability for the base
if p=0.01 (1% chance of error), then q=20
p = , (99.999% accuracy), q = 50
Phred quality values are rounded to the nearest integer

17. Main Illumina noise factors
Schematic representation of main Illumina noise factors.
(a–d) A DNA cluster comprises identical DNA templates (colored boxes) that are attached to the flow cell. Nascent strands (black boxes) and DNA polymerase (black ovals) are depicted.
(a) In the ideal situation, after several cycles the signal (green arrows) is strong, coherent and corresponds to the interrogated position.
(b) Phasing noise introduces lagging (blue arrows) and leading (red arrow) nascent strands, which transmit a mixture of signals.
(c) Fading is attributed to loss of material that reduces the signal intensity (c).
(d) Changes in the fluorophore cross-talk cause misinterpretation of the received signal (blue arrows; d). For simplicity, the noise factors are presented separately from each other.
Erlich et al. Nature Methods 5: (2008) 17

18. Comparison of existing methods
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, (2008)

19. Read length and pairing
ACTTAAGGCTGACTAGC
TCGTACCGATATGCTG
Short reads are problematic, because short sequences do not map uniquely to the genome.
Solution #1: Get longer reads.
Solution #2: Get paired reads.

20. Third generation Single-molecule sequencing Longer reads
no DNA amplification is involved
Helicos HeliScope
Pacific Biosciences SMRT …
Longer reads
Roche/454 > 400bp
Illumina/Solexa > 100bp
Pacific Bioscience > 1000 bp and single molecule

21. Applications of next-generation sequencing
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, (2008) 21

22. Analysis tasks Base calling Mapping to a reference genome
De novo or assisted genome assembly

23. References
Next-generation DNA sequencing, Shendure and Ji, Nat Biotechnol
Next-Generation DNA Sequencing Methods, Elaine R. Mardis, Annu. Rev. Genomics Hum. Genet. (2008) 9:387–402