Date post: | 31-May-2015 |
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Technology |
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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?