Contenu connexe Similaire à NGx Sequencing 101-platforms (20) NGx Sequencing 101-platforms4. Whole Genome Sequencing
30X coverage
90 Gb
Long reads
Paired-end
Mate pair
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
5. Exome Sequencing
100X+ coverage
5 Gb
Long reads
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
6. Small Genome Sequencing
1+ Gb
Long reads
Paired-end
multiplexing
Credit: Rocky Mountain Laboratories, NIAID, NIH Credit: Graham Colm (Wikipedia)
10. ChIP Sequencing
Lots of reads
multiplexing
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI)
17. Illumina – Performance Specs
HiSeq HiSeq
GAIIx HiScanSQ MiSeq
2000/2500 1000/1500
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 3B/600M 1.5B/300M 320M 750M 15M
reads/run
Instrument $690k/$74 $590k/$640 ~$300k ~$400k $125k
cost 0k k
Run cost ~$23k ~$11k ~$17k ~$11k ~$1k
18. Illumina – Applications
HiSeq HiSeq
GAIIx HiScanSQ MiSeq
2000/2500 1000/1500
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
19. Illumina – Future Developments
HiSeq HiSeq
GAIIx HiScanSQ MiSeq
2000/2500 1000/1500
Focus on desktop system
Longer reads
Push into diagnostics
Chemistry A = fast reads?
Chemistry B = long reads?
21. Life: SOLiD – Chemistry
Library Prep Amplification Sequencing
22. Life: SOLiD – Performance Specs
SOLiD SOLiD SOLiD
SOLiD 5500
5500xl 5500xl W 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 ~800M 2.4B ~400M 1.2B
reads/run
Instrument $595k $70k $349k $70k
cost upgrade upgrade
Run cost ~$10k ~$5k ~$5k ~$2.5k
23. Life: SOLiD – Applications
SOLiD SOLiD
5500xl(W) 5500(W)
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
24. Life: SOLiD – Future Developments
SOLiD SOLiD
5500xl(W) 5500(W)
Probably very little
Main focus is on Ion Torrent technology
27. 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 ~0.6M ~3M ~5.5M ~82M ~330M
reads/run
Instrument $50k $50k $50k $149k $149k
cost
Run cost $349 $549 $749 ~$1k ~$1k
28. 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
29. 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
32. 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 1M 0.1M
reads/run
Instrument ~$500k $125k
cost
Run cost ~$6k ~$1k
33. 454/Roche – Applications
GS FLX+ GS Jr.
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
34. 454/Roche – Future Developments
GS FLX+ GS Jr.
Probably very little
Roche has signaled the desire for new technology
37. Pacific Biosciences – Performance Specs
PacBio RS
‘C2’
Mb/run 120
Run time 40 min
Gb/day ~1 Gb
Read length 3kb (avg)
# of single ~50k
reads/run
Instrument ~$700k
cost
Run cost $100
38. Pacific Biosciences – Applications
PacBio RS
Whole Genome
Exome
Small Genome
Targeted
Transcriptome
RNA Profiling
ChIP-Seq
Metagenomics
39. Pacific Biosciences – Future Developments
PacBio RS
Longer reads
Reduced error rate
Increased chip density
Non-fluorescent detection?
43. Whole Genome Sequencing
ILMN HiSeq ✔
ILMN MiSeq
SOLiD ✔
Ion PGM
Ion Proton ✔
454 GS FLX+
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
✔ PacBio RS
44. Exome Sequencing
ILMN HiSeq ✔
ILMN MiSeq ✔
SOLiD ✔
Ion PGM
Ion Proton ✔
454 GS FLX+
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
PacBio RS
45. Small Genome Sequencing
ILMN HiSeq
ILMN MiSeq ✔
SOLiD
Ion PGM ✔
Ion Proton ✔
Credit: Rocky Mountain Laboratories, NIAID, NIH Credit: Graham Colm (Wikipedia)
454 GS FLX+ ✔
454 GS Jr.
✔
PacBio RS ✔
46. Targeted DNA Sequencing
ILMN HiSeq
ILMN MiSeq ✔
SOLiD
Ion PGM ✔
Ion Proton ✔
454 GS FLX+ ✔
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
✔
PacBio RS ✔
47. Transcriptome Sequencing
ILMN HiSeq ✔
ILMN MiSeq
SOLiD ✔
Ion PGM
Ion Proton ✔
454 GS FLX+
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
PacBio RS
48. RNA Profile Sequencing
ILMN HiSeq ✔
ILMN MiSeq
SOLiD ✔
Ion PGM
Ion Proton ✔
454 GS FLX+
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
PacBio RS
49. ChIP Sequencing
ILMN HiSeq
ILMN MiSeq
✔
SOLiD
Ion PGM
Ion Proton ✔
454 GS FLX+
Credits: Darryl Leja (NHGRI), Ian Dunham (EBI) 454 GS Jr.
PacBio RS
50. Metagenome Sequencing
ILMN HiSeq ✔
ILMN MiSeq
SOLiD ✔
Ion PGM
Ion Proton ✔
454 GS FLX+
454 GS Jr.
PacBio RS
52. Major Trends
Desktop machines
Ease of use
Faster runs
Diagnostic/clinical use
Longer reads
Nanopore technologies
54. 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?