Programming opentrons robot

This script automates a portion of the new opentrons pipetting robot using the opentrons api (OT-2 API V2). More specifically, it automates the dilution step (concentration normalisation) after PCR amplification and a picogreen assay in the arctic protocol on all samples above the threshold concentration. It then performs the end-repair and barcoding on all normalized samples. Samples are then consolidated into a single tube.

Input data

This protocol accepts raw DNA concentration data from the Optima plate reader in .xlsx format. The most recent .xlsx file will be read in from /path/on/robot/ to serve as an entry point to the script. Upload an .xlsx file relating to the samples to run the script.

Info

• Save plate reader output as a .xlsx file in /path/on/robot/
• The samples will need to be laid out as described in the SARS_SOP.
• Up to 24 samples per run.
• Samples with a concentration < 4ng/ul will be skipped.
• The position of the sample on the plate will determine which barcode is used.
• Columns 1,2 and 3 can contain sample data. Column 4 must contain Pico standards data.

Example of a plate layout of 21 samples and Pico green standards, with optima data in cells:

	Sample	Sample	Sample	Pico
	1	2	3	4	5	6	7	8	9	10	11	12
A	677	28594	3233	121
B	26822	27726	43193	43244
C	41125	1523	19965	5330
D	7455	42273	2204	709
E	38978	43020	312	186
F	3312	6831
G	1476	44124
H	1650	2212

Simulate protocol

Create and activate a conda environment to get the minimum python version required for a non-jupyter opentrons installation

conda env create -f opentrons_env.yml
conda activate opentrons_env

Simulate the protocol from the command line

opentrons_simulate normalisation.py

Deck lay out

This protocol requires:

• Tips:

  1. Opentrons 96 filtertips 20ul; Slots 8 and 9
  2. Opentrons 96 filtertips 300ul; Slot 6

• Temperature Module: Slot 10

• 12 well reservoir 22ml: Slot 11

• 24 tube rack for enzymes: Slots 4 and 7

• End repair kit

• Ligation kit

• Barcodes

Barcodes rack layout

Barcodes are in held in a 24 tube rack in slot 4. The tubes should be laid out as shown below.

	1	2	3	4	5	6
A	NB01	NB02	NB03	NB04	NB05	NB06
B	NB07	NB08	NB09	NB10	NB11	NB12
C	NB13	NB14	NB15	NB16	NB17	NB18
D	NB19	NB20	NB21	NB22	NB23	NB24

Enzyme rack layout

	1	2	3	4	5	6
A	End repair Buffer 5x	End repair Enzyme	Empty Tube	Ligation Master Mix	Ligation Enhancer	Empty Tube
B
C
D	Empty Tube

Empty tubes are for making master-mixes and collection of the final samples. It is very important these are included or you will lose all the samples and waste the reagents.