The zenodo and megatests things are nf-core specific so we will need to request those

Hey @Adam Taylor we have a PR to main that only changes the changelog for the upcoming release, do you think it is ok for you and Jeremy to ok it or do we need to go to review-exchange?

2.0.0 is now live! Thanks to everyone who contributed. https://nf-co.re/mcmicro/2.0.0/

Awesome work everyone! 👏 Congratulations!

I think this story would make a great blog post if anyone fancies it 🤩

@Adam Taylor Updating your PR that removes ext.when for the segmenters so it's mergeable. Linting uncovered that you edited the local copy of the cellpose and mesmer modules without creating a

.diff

. I'm looking at the most recent upstream versions of those modules and it looks like cellpose added

process_gpu

so maybe we should just update for that. Can you explain what was fixed by adding the

stageAs

arg to

path(model, stageAs: '?/*')

?

I'm going to back out those changes for now and merge the other stuff. Then we can update the modules and see what remains to either patch locally or change upstream.

MCCELLPOSE fails on coreograph outputs as it receives flat tiff outputs but requires ome-tiffs with pixel size. Old issue here, but bumping into it as I use v2. I think the fix should be in coreograph to output OME-TIFF, or we could drop the input read conditions to mccellpose but that feels less clean

Finally cornered one of the more insidious bugs I've hit in a while in nf-core/mcmicro. Multi-sample runs die in MCQUANT with "the number of channels in markers.csv doesn't match the image." It cost me a long detour before the real culprit turned up. For multi-sample runs, mcmicro

collectFiles marker_name

across all samples into one shared

markers.csv

(with no per-sample key) and hands that same frankensheet to every sample's

MCQUANT

, so the marker count (64 in my run) never matches a single image's 44. Single-sample runs pass because there's only one sample's markers to pile up, which is also why it's slipped through: the test datasets are all single-sample. Fix is smallish: build the marker file per sample, keyed by

meta.id

. I can make a PR

Hello @Jeremy Muhlich, I made a small script to convert from MACSima RawData output to MCMICRO ometif input

I am using the macsima2mc script from github.com/SchapiroLabor/macsima2mc

My current 2.mcmicro_macsima2mc.sh script still uses the MCMICRO v1.0, and not the new nf-core version. Are all the modules from the previous version (UMAP, dendrogram, Scimap) available in the new nf-core version?

No, we're still fixing a few post-release issues we discovered since launching v2.0.0

Also I see the module

mcstaging/macsima2mc

made it into nf-core modules earlier this year. Maybe someone is working on integrating that into nf-core/mcmicro now.

Great, thank you! and amazing work, very thankful for this tool

Thanks to @Carolin Schwitalla and @EfraMP for the nf-core module work

I am currently trying out the module and getting familiar with MACSima data and the subsequent tools. But happy to help or work on an integration to mcmicro.

Hello, I have a question, Macsima structures its output per cycle, and Ashlar takes an input folder containing images from all cycles and registers them. Will this create 1 image file with all the channels as a stack?

Hey, I was doing investigation on a tool called MONAI using Gemini's Deep Research tool. It mentioned this workflow as a workflow that could benefit from it docs.google.com/document/d/1H0jhd0whoQN…/edit?usp=sharing

Hello, I have been working with some labs that do MACSima experiments on many different markers, and they would split the experiment across separate runs. I made a nextflow script for appending multiple runs into a single RawData folder that can be then used with the staging script (macsima2mc) and the mcmicro pipeline. Feel free to take any of these and adjust if needed: github.com/StonyBrookRCI/MACSIMA_MCMICRO/tree/main A separate issue we had was that some groups used single FOV experiments which causes errors during the illumination correction step. I also want to improve the staging script to dynamically detect ROIs with a single FOV per cycle to turn off illumination correction, or leave it on when multiple FOVs are present.

Are they combining single-FOV experiments with many-FOV experiments in the same sample sheet for mcmicro? We only support turning illumination correction on/off for the entire sample sheet, not per sample.

They aren't combinining single FOV with many FOV, usually they would run a single FOV on experiments for spheroids with many markers and multiple runs, and multiple FOV on larger tissues with smaller panels

Would it be possible to add to the nf-core documentation a samplesheet example with background substraction columns? Currently is a bit hidden under the backsub module

Does anyone want to do a quick review on a minor tools update? https://github.com/nf-core/mcmicro/pull/172 I want to resolve the conflicts in this one before moving on to merge+resolve the tools v4.1.0 update.

Never mind, traded a review with another pipeline

Hello, i would work on adding macsima staging to mcmicro. And since it would add additional input logic i wanted to align with you on how this would be added. I tested different sample sheet options for staging Macsima images with macsima2mc: • define one ROI per sample sheet row • define one experiment per sample sheet row ( with multiple ROIs) From the feedback of people who process their datasets with mcmicro, they would like to reduce the sample sheet so they dont need to define 40 ROIs, for example, for a project. Yes, let me know what you think, i can start working on a PR. Maybe it’s easier to discuss details of the code there. 🙂

Hello Carolin, I made a staging.nf script to take the RawData from MACSima and generate the ome.tifs and samplesheet for the mcmicro pipeline. I would need help adjusting the code to run it as a container instead of on my conda environment. It currently uses the package developed by the Schapiro Lab

1.staging.nf

@Alejandro Gil Gomez btw, if you use the macsima2mc staging tool for processing already, update the tool to the newest version. There was a bug that mismatched the exposure time for the channels in the markersheet, which affects the background subtraction. I think the effects were not major, but there were visible differences for some markers.