High Throughput Small Molecule Detection - Skyline · 2017-06-06 · High Throughput Small Molecule...

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