Oct 22, 2025
Version 2
High-throughput workflow for the genotypic characterization of transposon insertion library variants V.2
- Lorea Alejaldre1,2,
- Ana Mariya Anhel2,
- Lewis Grozinger1,2,
- Ángel oñi-Moreno1,2
- 1Centro Nacional de Biotecnologia (CNB-CSIC) Madrid, Spain;
- 2Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain
- Lorea Alejaldre: [email protected];
- Ángel oñi-Moreno: [email protected]
Protocol Citation: Lorea Alejaldre, Ana Mariya Anhel, Lewis Grozinger, Ángel oñi-Moreno 2025. High-throughput workflow for the genotypic characterization of transposon insertion library variants. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg394jzg25/v2Version created by Lorea Alejaldre
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 09, 2024
Last Modified: October 22, 2025
Protocol Integer ID: 93149
Keywords: High-throughput, Transposon library, marC9, Tn5, SEVA, OT-2, Opentrons, genotypic characterization, genotyping, bacterial genome, 96-well, characterization of transposon library variant, transposon insertion library variant, transposon library variant, throughput workflow for the genotypic characterization, blastn for genomic annotation, transposon insertion site, workflow for the genotypic characterization, genomic read, other transposon system, genomic annotation, genotypic characterization, plasmid set, pcr sample preparation, sib pbamd1, ps6 primer, genotyping, well plate format
Funders Acknowledgements:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555,2019-T1/BIO-14053
MCIN/AEI
Grant ID: PID2020-117205GA-I00, CNS2022-135951, PID2023-152470NB-I00
ERC
Grant ID: 101044360
Abstract
This is a workflow for the genotypic characterization of transposon library variants. It has been developed using an open-source Opentrons OT-2 robot, BLASTN for genomic annotations and modular sub-protocols (e.g., PCR sample preparation, OT-2 volume transfer, OT-2 counter selection, etc) that can be used for other tasks, thus providing a general-purpose pipeline.
All steps follow a 96-well plate format for high-throughput analysis. The protocol is described for the characterization of transposon library variants generated with SEVA-Sib pBAMD1-x and pBLAM1-x plasmid sets that follow Standard European Vector Architecture (SEVA, https://seva-plasmids.com) and can be amplified with the standard PS1-PS6 primers. However, it can be adapted for other transposon systems.
Changes from previous version:
- This version references updated OT-2 automation protocols which now follow the standardized LAP format (https://www.laprepo.com).
- Genotyping is performed with a new ad hoc python package to annotate genomic reads and identify the transposon insertion sites.
Guidelines
This workflow comprises the following sections: 1) Colony picking in selective media 2) Counter-selection and glycerol stocks pre-cultures 3) Colony selection in OT-2 liquid handler robot 4) Master 96-well plate for PCR steps 5) Control PCRs (spurious plasmid integration control and cargo insertion control) 5) Arbitrary PCRs 6) Sequencing and annotation. There is an additional section with an example on how to run the script.
We recommend the use of an OT-2 protocol specially if more than 2 libraries are to be analyzed. However, we recommend to do counter-selection in the OT-2 liquid handling robot even for one plate to avoid human errors.
Note that other pipettes can be used to run the workflow in the OT-2 but these were deemed the most appropriate for the overall workflow to minimize pipette changes.
Materials
Equipment:
Equipment
Incubating mini-shaker
NAME
Incubating shaker
TYPE
Fisherbrand
BRAND
15554070
SKU
LINK
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Equipment
SPECTROstar Nano
NAME
plate reader
TYPE
BMG
BRAND
SPECTROstar Nano
SKU
LINK
Equipment
Mastercycler® nexus - PCR Thermal Cycler
NAME
Thermocycler
TYPE
Eppendorf
BRAND
12304943
SKU
LINK
Equipment
Centrifuge Tube Mini-Cooler
NAME
Cold Block
TYPE
BRAND
BRAND
10141921
SKU
LINK
Electrophoresis machine
Wet-lab requirements:
Material
- Non-treated flat bottom sterile 96-well plates 96-well plates flat bottom non-treatedVWR International (Avantor)Catalog #734-2781
- Sterile breathable membrane for 96-well plate Greiner Bio-One Sellador BREATHseal™Fisher ScientificCatalog #11920667
- Storage membrane for 96-well plate Thermo Scientific™ Láminas de papel de aluminio adhesivas para placas de PCRFisher ScientificCatalog #10130853
- PCR plates
Thermo Scientific™ PCR Plate, 96-well, low profile, skirted, redFisher ScientificCatalog #10161073
Enzymes
- DNA polymerase with green buffer
Phire Green Hot Start II PCR Master MixFisher ScientificCatalog #15391732
- DNA polymerase
Thermo Scientific™ Phire Hot Start II PCR Master MixFisher ScientificCatalog #15361732
Oligonucleotides:
- Arbitrary PCR: ARB2, ME-O-Km-Ext-F, ME-O-Km-Int-F, ME-O-Sm-Ext-F, ME-O-Sm-Int-F, ME-O-Gm-Ext-F, ME-O-Gm-Ext-R (https://doi.org/10.3389/fbioe.2014.00046)
- Optional insert control: PSMCS
Dry-lab requirements:
- Python 3
- Command-line BLASTN
Colony picking in selective media
1d
Dispense 100 µL of selective media (M9-citrate for P. putida or Luria-Bertani plus 20 ng/µL nalidixic acid for DH5α E. coli) plus transposon cassette antiobiotic in a 96-well plate
Pick individual colonies into a 96-well plate with selective media
Tip: Keep tips inside of wells to keep track
Cover with a sterile breathable membrane
Grow Overnight at 30 °C (P. putida) or 37 °C (E. coli) / 500 rpm
Counter-selection and glycerol stocks pre-cultures
1d
Measure OD600nm of overnight culture grown in selective media from plus transposon cassette antibiotic in a plate reader
Inoculation of counter-selection plate in selective media:
- Dispense 100 µL of selective media (M9-citrate for P. putida or Luria-Bertani plus 20 ng/µL nalidixic acid for DH5α E. coli) plus ampicillin (backbone antibiotic) to select against spurious integration events.
- Transfer 5 µL of overnight culture from to counter-selection (ampicillin) plate
- Cover with a sterile breathable membrane
Note
For steps 6 and 7, if two or more 96-well plates are used as input it is advised to use the OT-2 protocol below to minimize human error. Dispensed volume and culture volume inoculated should be that described in these steps. Note that steps 6 and 7 could be completed together in a single run depending on the number of initial plates.
10m
Inoculation of precultures for glycerol stock in rich media:
- Dispense 100 µL of Luria-Bertani media plus transposon cassette antiobiotic in a 96-well plate
- Transfer 5 µL of overnight culture from to counter-selection (ampicillin) plate
- Cover with a sterile breathable membrane
10m
Grow counter-selection and glycerol stock pre-culture plates Overnight at 30 °C (P. putida) or 37 °C (E. coli) / 500 rpm
16h
Measure OD600nm of overnight culture grown in selective media plus ampicillin in a plate reader
Colony selection in OT-2 liquid handler robot
- Selection of colonies to store as glycerol stocks and do further PCR reactions by running the following OT-2 protocol:
Note
The OT-2 protocol will prepare three plates (2 glycerol stock plates and a "PCR plate") and perform the following:
- Dispense 75 µL of PCR-grade water to "PCR plate"
- Dispense 25 µL of 30% glycerol to two glycerol stock plates
- Transfer 25 µL of grown pre culture in Luria-Bertani media plus transposon cassette antibiotic from to "PCR plate" and glycerol stock plates
Cover glycerol stock plates with a storage membrane and store at -80ºC
If not proceeding to the next step right away: Store "PCR plate" at 4ºC for a few days or cover with an storage membrane and store at -20ºC for longer term
Master 96-well plate for PCR steps
Transfer 50 µL of selected colonies from one or more libraries to a 96-well plate with the following OT-2 protocol:
Protocol
CREATED BY
Biocomputation Lab
Control PCRs
Safety information
Positive control (donor plasmid) and wild-type control (P. putida or E. coli) should be added to every reaction in this section.
Spurious integration control with SEVA primers pairs PS3/PS4 and PS5/PS6
Note
If <48 cfu are to be analyzed both PS3/PS4 and PS5/PS6 spurious integration controls can be done in a single 96-well plate
Optional: Cargo integration control with primers PSMCS and either ME-O-Km-R/ME-O-Sm-R or ME-O-Gm-R (depending on transposon cassette antibiotic)
Arbitrary PCRs
Arbitrary PCR#1 using primer pairs ARB6 and ME-O-Km-Ext-F/ME-O-Sm-Ext-F or ME-O-Gm-Ext-F depending on transposon antibiotic cassette
Note
The OT-2 protocol will perform the following steps:
- Prepare a PCR master mix
- Dispense 19 µL of PCR mastermix
- Transfer 2 µL of pre-culture from
Seal 96-well plate, place it in thermocycler and run the following PCR program:
| A | B | C | |
| 98ºC | 5 min | ||
| 98ºC | 10 s | x6 cycles | |
| 30ºC | 30 s | ||
| 72ºC | 1 min 30 s | ||
| 98ºC | 10 s | x30 cycles | |
| 45ºC | 30 s | ||
| 72ºC | 1 min 30 s | ||
| 72 ºC | 5 min | ||
| 4ºC | hold |
Select 8-12 Arbitrary PCR#1 reactions from the 96-well plate and run them on a 1% agarose gel to verify amplification.
Note
Several bands will appear and even DNA smears even when the reaction has worked perfectly.
Arbitrary PCR#2 using primers pairs ARB2 and ME-O-Km-Int-F/ME-O-Sm-Int-F or ME-O-Gm-Int-F, depending on transposon antibiotic cassette, run the following OT-2 protocol:
Note
The OT-2 protocol will perform the following steps:
- Prepare a PCR master mix
- Transfer 1 µL of PCR product from Arbitrary PCR#1
- Dispense 19 µL of PCR mastermix
Seal 96-well plate, place it in thermocycler and run the following PCR program:
| A | B | C | |
| 98ºC | 30 s | ||
| 98ªC | 10 s | x 30 cycles | |
| 52ºC | 30 s | ||
| 72ºC | 1 min 30 s | ||
| 72ºC | 5 min | ||
| 4ºC | hold |
Select 8-12 Arbitrary PCR#2 reactions from the 96-well plate and run them on a 1% agarose gel to verify amplification
Note
Several bands will appear and even DNA smears even when the reaction has worked perfectly.
Sequencing and annotation
1m
Prepare a PCR plate to send to sequencing by mixing 10 µL of unpurified Arbitrary PCR#2 reaction and 10 µL of 10 µM sequencing primer (ME-O-Km-Ext-F/ME-O-Sm-Ext-F or ME-O-Gm-Ext-F depending on the transposon antibiotic cassette)
Note
These guidelines may vary depending on the sequencing service arranged for your laboratory.
Annotate sequencing results by running the following protocol:
Note
The python package uses as input:
- DNA sequencing results in .txt or .seq
- Reference genome file in GENBANK format
- Transposon sequence file in GENBANK format
Optional:
1. .ab1 files to verify sequencing quality
2. map layout from previous steps
1m