ot-flex-automation

Opentrons Flex automation for genomics library preparation. The repository contains whole-genome sequencing, methylation sequencing, and TIP-seq workflows plus a small orchestration layer for closed-loop quality control.

Each protocol keeps liquid-handling actions on the robot and presents thermal cycling, fragmentation, magnet moves, and instrument-based QC as explicit operator handoffs.

The public WGS and methylation files are water-only motion profiles. Their uniform synthetic volumes exist solely to exercise deck choreography and are not wet-lab instructions. Biological execution requires an uncommitted OPERATOR_METHOD_PROFILE containing locally validated volumes, programs, cleanup rules, and QC criteria.

Protocols

File	Workflow	Output	Status
protocols/whole_genome_seq_flex.py	Whole-genome sequencing preparation	Whole-genome libraries	Deck choreography run end to end on the Flex; public profile is water only
protocols/methylation_sequencing_flex.py	Methylation sequencing	Genome-wide methylation libraries	Draft; motion-test ready
protocols/tipseq_epigenome_flex.py	TIP-seq epigenomic profiling	Histone-mark and transcription-factor libraries	Draft; motion-test ready

All protocols use protocol.pause() for off-deck steps. WGS and methylation expose a private-profile seam near the top of the file; their committed fallback contains water-only stage labels and no biological parameter defaults.

Whole-genome sequencing

The WGS choreography models these functional boundaries:

1. Input preparation and genome amplification.
2. Library construction and PCR enrichment.
3. Profile-defined cleanup, elution, and output transfer.

The public profile maps those boundaries to equal water transfers. It does not publish reagent composition, stage-specific volume, thermal program, cleanup ratio, or QC threshold.

Default deck:

• Sample plate: B2
• Reagent reservoir: B3
• Magnetic block: C2
• Output plate: C3
• Bead and wash reservoir: D2
• Tip racks: A2 and A3
• Trash: D1

The public B3 map uses A1 input preparation, A2 genome amplification, A3 library construction, and A4 PCR enrichment. D2 uses A1 cleanup water, A2 wash water, A3 elution water, and A12 waste.

Methylation sequencing

The methylation choreography provides configurable library-construction, methylation-processing, PCR-enrichment, cleanup, and QC boundaries. It does not select a specific methylation chemistry. Two plates alternate through the synthetic cleanup motion so the deck behavior remains testable.

The public B3 map uses water in A1-A3 for the three synthetic stages. A private operator profile may define different stage wells and cleanup choices.

TIP-seq

The TIP-seq workflow preserves the published Tn5, in-vitro transcription, and bead carry-through structure of the assay. Upstream cell binding, antibody incubation, transposase binding, and washes remain manual. The robot handles tagmentation onward, including master-mix additions and SPRI cleanups.

Pre- and post-cleanup DNA-quantification checkpoints are represented as pauses. TIP-seq uses three tip racks plus one mid-run refill for a single column.

Hardware and consumables

• Opentrons Flex with an 8-channel 1000 µL pipette
• Opentrons Magnetic Block GEN1
• 12-well reservoirs
• 96-well PCR plates
• 200 µL filter-tip racks
• External thermal cycler
• Fluorometric DNA quantification and fragment-analysis instruments
• Off-deck fragmentation equipment where required

Running a protocol

The committed WGS and methylation profiles are one-column motion tests (NUM_SAMPLES = 8).

• Load water only in the mapped stage and cleanup wells.
• Import the protocol into the Opentrons App and complete Labware Position Check.
• For biological work, create a private copy and populate OPERATOR_METHOD_PROFILE; the public fallback must not be used with samples or reagents.

Each profile is validated for required keys and safe pipette-volume bounds before movement. Off-deck timing, thermal programs, chemistry, and acceptance criteria remain operator-supplied.

Portable orchestration

orchestration/portable/ represents transfer intent independently of a robot API. Public examples render synthetic water choreography; a controlled local spec is required for biological method values.

orchestration/ also includes:

• A concentration-based QC decision engine
• A CSV fluorescence-plate-reader adapter
• A Flex HTTP link
• A camera-based bead-pellet QC interface

These components are scaffolding for supervised, closed-loop runs. WGS decision logic requires a caller-supplied WgsQcProfile; the repository defines no default yield, fragment, or normalization target.

Alternate-deck WGS variants

The alternate-deck WGS files use the Flex 8-channel 1000 µL pipette on the left mount and lower source aspiration in B3 and D2:

File	Use
protocols/whole_genome_seq_full_8ch_returntip_lower_source_demo.py	Water-only demo that returns one reused tip column
protocols/whole_genome_seq_full_flex_v2_lower_source.py	Fresh-tip motion profile with a private-profile seam

The return-tip file is hard-limited to water. The fresh-tip file still defaults to water and requires an uncommitted validated profile for biological work.

References

• Bartlett et al. (2021), TIP-seq
• Kaya-Okur et al. (2019), CUT&Tag
• Opentrons Python Protocol API