CS 6293 Advanced Topics:
Bioinformatics
Next-generation sequencing technology
Outline
• First generation sequencing
• Next generation sequencing (current)
– AKA:
• Second generation sequencing
• Massively parallel sequencing
• Ultra high-throughput sequencing
• Future generation sequencing
• Analysis challenges
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, 1135 - 1145 (2008)
Cyclic-array methods (nextgeneration)
• 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, 1135 - 1145 (2008)
Available next-generation
sequencing platforms
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Illumina/Solexa
ABI SOLiD
Roche 454
Polonator
HeliScope
…
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
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
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.
http://www.illumina.com/pages.ilmn?ID=203
First Round
All 4 labeled nucleotides
Primers
Polymerase
1. Take image of first cycle
2. Remove fluorophore
3. Remove block on 3’ terminus
Stats:
• read lengths up to 36 bp
• error rates 1-1.5%
• several million “spots” per lane (8 lanes)
• cost $2 per megabase
http://seq.molbiol.ru/
Conventional sequencing
• Can sequence up to 1,000 bp, and perbase 'raw' accuracies as high as 99.999%.
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, 1135 - 1145 (2008)
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
Phred Quality Score
q  10log10 ( p)
•
•
•
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p=error probability for the base
if p=0.01 (1% chance of error), then q=20
p = 0.00001, (99.999% accuracy), q = 50
Phred quality values are rounded to the
nearest integer
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: 679-682 (2008)
Comparison of existing methods
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, 1135 - 1145 (2008)
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.
Third generation
• Single-molecule sequencing
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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
Applications of next-generation
sequencing
Jay Shendure & Hanlee Ji, Nature Biotechnology 26, 1135 - 1145 (2008)
Analysis tasks
• Base calling
• Mapping to a reference genome
• De novo or assisted genome assembly
References
• Next-generation DNA sequencing,
Shendure and Ji, Nat Biotechnol. 2008.
• Next-Generation DNA Sequencing
Methods, Elaine R. Mardis, Annu. Rev.
Genomics Hum. Genet. (2008) 9:387–402