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The field of next-generation sequencing (NGS) has been experiencing explosive growth over the past several years and shows little sign of slowing down. The increasing capabilities and dramatically lowered costs have expanded NGS's reach beyond that of the human genome into nearly every corner of biological research. An overview of the platforms on the market today, including an assessment of their relative strengths and weaknesses, will be presented. The presentation will conclude with a peek into where the technology is going and what will be available in the future.
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Shawn C. Baker, Ph.D.
Sequencing 101 – NGS Platforms
Overview
Review Major Applications
Review Major Platforms
Future Trends
Asking the Right Questions
Applications
Whole Genome Sequencing
30X coverage
90 Gb
Long reads
Paired-end
Mate pair
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
Exome Sequencing
100X+ coverage
5 Gb
Long reads
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
Small Genome Sequencing
1+ Gb
Long reads
Paired-end
multiplexing
Credit: Rocky Mountain Laboratories, NIAID, NIH
Credit: Graham Colm (Wikipedia)
Targeted DNA Sequencing
10+ Mb
multiplexing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
Transcriptome Sequencing
Lots of reads
Long reads
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
RNA Profile Sequencing
Lots of reads
multiplexing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ChIP Sequencing
Lots of reads
multiplexing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
Metagenome Sequencing
Lots of reads
Long reads
Paired-end
Applications
Platforms
Platforms
Platforms
Illumina – Chemistry
Library Prep
Amplification
Sequencing
Illumina – Performance Specs
HiSeq 2000/25
00
HiSeq 1000/15
00GAIIx
HiScanSQ
MiSeq
Gb/run 600/120 300/60 95 150 7
Run time 11d/27hrs
8.5d/27hrs
14d 8.5d 35hrs
Gb/day 55/120 35/60 ~7 18 ~7
Read length
2x100/150
2x100/150
2x150 2x100 2x250
# of single reads/run
3B/600M 1.5B/300M
320M 750M 15M
Instrument cost
$690k/$740k
$590k/$640k
~$300k ~$400k $125k
Run cost ~$23k ~$11k ~$17k ~$11k ~$1k
Illumina – Applications
HiSeq 2000/25
00
HiSeq 1000/15
00GAIIx
HiScanSQ
MiSeq
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
Illumina – Future Developments
HiSeq 2000/25
00
HiSeq 1000/15
00GAIIx
HiScanSQ
MiSeq
Focus on desktop system Longer reads Push into diagnostics Chemistry A = fast reads? Chemistry B = long reads?
Platforms
Life: SOLiD – Chemistry
Library Prep
Amplification
Sequencing
Life: SOLiD – Performance Specs
SOLiD 5500xl
SOLiD 5500xl
W
SOLiD 5500
SOLiD 5500 W
Gb/run 95 240 48 120
Run time 6 days 10 days 6 days 10 days
Gb/day ~16 24 ~8 12
Read length
2X60 2X50 2X60 2X50
# of single reads/run
~800M 2.4B ~400M 1.2B
Instrument cost
$595k $70k upgrade
$349k $70k upgrade
Run cost ~$10k ~$5k ~$5k ~$2.5k
Life: SOLiD – Applications
SOLiD 5500xl(
W)
SOLiD 5500(W)
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
Life: SOLiD – Future Developments
SOLiD 5500xl(
W)
SOLiD 5500(W)
Probably very little Main focus is on Ion Torrent technology
Platforms
Life: Ion Torrent – Chemistry
Library Prep
Amplification
Sequencing
Life: Ion Torrent – Performance Specs
PGM 314
PGM 316
PGM 318
Proton 1
Proton 2
Gb/run 10-40 Mb 100-400 Mb
1 Gb ~10 Gb ~100 Gb
Run time 2 hours 2 hours 2 hours ~4 hours ~4 hours
Gb/day ~120 Mb ~1.2 Gb ~3 Gb ~30 Gb ~ 300 Gb
Read length
200b 200b 200b 200b >200b
# of single reads/run
~0.6M ~3M ~5.5M ~82M ~330M
Instrument cost
$50k $50k $50k $149k $149k
Run cost $349 $549 $749 ~$1k ~$1k
Life: Ion Torrent – Applications
PGM 314
PGM 316 PGM 318 Proton 1 Proton 2
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
Life: Ion Torrent – Future Developments
PGM 314
PGM 316 PGM 318 Proton 1 Proton 2
Focus on desktop systems Longer reads Increased chip densities New machine? Push into diagnostics
Platforms
454/Roche – Chemistry
Library Prep
Amplification
Sequencing
454/Roche – Performance Specs
GS FLX+
GS Jr.
Mb/run 700 35
Run time 23 hours 10 hours
Mb/day 700 35
Read length
Up to 1kb ~400b
# of single reads/run
1M 0.1M
Instrument cost
~$500k $125k
Run cost ~$6k ~$1k
454/Roche – Applications
GS FLX+
GS Jr.
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
454/Roche – Future Developments
GS FLX+
GS Jr.
Probably very little Roche has signaled the desire for new technology
Platforms
Pacific Biosciences – Chemistry
Library Prep
Amplification
Sequencing
Pacific Biosciences – Performance Specs
PacBio RS
‘C2’
Mb/run 120
Run time 40 min
Gb/day ~1 Gb
Read length
3kb (avg)
# of single reads/run
~50k
Instrument cost
~$700k
Run cost $100
Pacific Biosciences – Applications
PacBio RS
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
Pacific Biosciences – Future Developments
PacBio RS
Longer reads Reduced error rate Increased chip density Non-fluorescent detection?
Other Platforms…
More Information…
www.blueseq.com
Matching Applications with Platforms
Whole Genome Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
✔
Exome Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
✔
Small Genome Sequencing
Credit: Rocky Mountain Laboratories, NIAID, NIH
Credit: Graham Colm (Wikipedia)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
✔
✔
✔
Targeted DNA Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
✔
✔
✔
Transcriptome Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
RNA Profile Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
ChIP Sequencing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
Metagenome Sequencing
ILMN HiSeq
ILMN MiSeq
SOLiD
Ion PGM
Ion Proton
454 GS FLX+
454 GS Jr.
PacBio RS
✔
✔
✔
Major Trends
Major Trends
Desktop machines
Ease of use
Faster runs
Diagnostic/clinical use
Longer reads
Nanopore technologies
Asking the Right Questions
Asking the Right Questions
Before you buy a platform Do I need to buy a platform? What are my major applications? What are my colleagues using?
Before you start sequencing What am I trying to answer? How will I analyze the data? Where is my variation coming from? How many samples do I need?
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