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High Throughput Small Molecule DetectionSkyline User Group Meeting 6/4/2017
Yang Zhang
2 Copyright © 2017 Amyris, Inc. All rights reserved
Amyris: from sugar to product
FARNESENE (C15)
SUGARSOURCE
MONOTERPENES (C10)
ISOPRENE (C5)
ARTEMISININANTI-MALARIAL (C15)
3 Copyright © 2017 Amyris, Inc. All rights reserved
DARPA M2K: from mg to kg in record time
Year 1 Attempt to
produce 450 molecules at
mg scale
Year 2150
molecules at gm scale
Year 350 molecules at g/L/h scale
Year 4Purify 1 Kg of 10 molecules
reduce the cost of scale up by 10X
Cheaper
increase molecule throughput by 20X
Greater
reduce the time to market by 4X
Faster
4 Copyright © 2017 Amyris, Inc. All rights reserved
Analytical workflow
Data Acquisition Data analysis• Peak integration• Peak alignment• Hit calling• Titer estimation
Data uploading• Generate reports• Save to database
Thousands of samples per
week covering 500+ molecules
5 Copyright © 2017 Amyris, Inc. All rights reserved
Peak integration without RT correction
Skyline picked the best peak at 4 minutes
6 Copyright © 2017 Amyris, Inc. All rights reserved
Get RT from standard or best guess in sample
Standard peak showsat 3.5 minute
7 Copyright © 2017 Amyris, Inc. All rights reserved
Correct RT
Correct peak to 3.4 minute
8 Copyright © 2017 Amyris, Inc. All rights reserved
Skylinerunner + R: naïve peak integration
SkylineRunner.exe --in=skyline.sky--import-transition-list=transition.csv --out=skyline.sky
SkylineRunner.exe --in=skyline.sky –report-name=M2K--report-file=M2K.tsv --report-format=tsv--report-invariant --report-add=M2K.skyr --report-conflict-resolution=overwrite
9 Copyright © 2017 Amyris, Inc. All rights reserved
Skylinerunner + R: Retention time correction
SkylineRunner.exe --in=skyline.sky--import-transition-list=transition.csv --out=skyline.sky
SkylineRunner.exe --in=skyline.sky –report-name=M2K--report-file=M2K.tsv --report-format=tsv--report-invariant --report-add=M2K.skyr --report-conflict-resolution=overwrite
10 Copyright © 2017 Amyris, Inc. All rights reserved
Example output
Sample
Control
Blank
11 Copyright © 2017 Amyris, Inc. All rights reserved
Performance comparable to manual curation
12 Copyright © 2017 Amyris, Inc. All rights reserved
Hands-on time dropped from hours to minutes
Before:
Now:
13 Copyright © 2017 Amyris, Inc. All rights reserved
Acknowledgement