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two-photon

This repository contains utilities for analyzing 2p data:

Analysis Pipeline

The analysis pipeline consists of the following stages:

  • raw2tiff: converts Bruker proprietary output format to a TIFF stack
  • convert: converts tiff and csv/text files to hdf5.
  • preprocess: detect and remove stim artefacts
  • qa: make QA plots to check stim artefact removal
  • analyze: run suite2p, optionally combining multiple preprocessed datasets
  • backup: back up input/intermediate/output data to a safe place

Setup

First, install the code. You can use GitHub desktop, or use git on the command line. This only has to be done once.

git clone https://github.com/deisseroth-lab/two-photon.git

Next, install the environment. You will need to install conda first. Then use the following command from within the directory where you installed the repo above. This also only has to be done once.

conda env create -f environment.yml -n two-photon
conda activate two-photon
pip install -e .  # installs the 2p script (in editable mode, so you can update the code)

Executing

To run the processing script, the environment needs to be activated. This needs to be done each time you start a new terminal.

conda activate two-photon

The executable is called 2p, and each stage is a different subcommand that can be run. It is possible to run multiple stages by specifying multiple subcommands.

Data Layout and Global Flags

The scripts required a strict layout of data, and assume the input data follows a directory structure and filenaming that the Bruker scopes create. The data is setup in subdirectories of a base-path, named by the stage and the acquisition name.

To point the script to the correct location of of dataset, use the following flags:

  --base-path PATH    Top-level storage for local data.  [required]
  --acquisition TEXT  Acquisition sub-directory to process.  [required]

Using the following global flags (meaning after 2p but before other commands or flags):

2p \
    --base-path /my/data \
    --acquisition 20210428M198/slm-001

will use the following locations for the data. Note the expected location of the raw data.

data type location
RAWDATA, csv, xml, and env files from scope /my/data/raw/20210428M198/slm-001
tiff stacks /my/data/tiff/20210428M198/slm-001
converted hdf5 data /my/data/convert/20210428M198/slm-001
preprocess /my/data/preprocess/20210428M198/slm-001
qa /my/data/qa/20210428M198/slm-001
analyze - suite2p output /my/data/analyze/20210428M198/slm-001

Command: raw2tiff

The raw2tiff command runs the Bruker software to rip the RAWDATA into a tiff stack. This is a Windows-only command, until the kinks of running on Linux are ironed out.

Example:

2p \
    --base-path /my/data \
    --acquisition 20210428M198/slm-001
    raw2tiff

Command: convert

The convert command converts the tiff stacks and voltage data to hdf5.

Example:

2p \
    --base-path /my/data \
    --acquisition 20210428M198/slm-001 \
    convert --channel 3

Command: preprocess

The preprocess command performs processing like stim removal on the data. It should be run even if there are no stim artefacts (in which case, no actual computation is done), so that downstream stages find the data in the correct place.

Example:

2p \
    --base-path /my/data \
    --acquisition 20210428M198/slm-001 \
    preprocess --frame-channel-name="frame starts" --stim-channel-name=respir

Example based on piezeo period:

2p \
    --base-path /media/hdd0/two-photon/minoue2/2p_CNC/ \
    --acquisition Chris_210429/10263_920nm_PC250-300-001  \
    preprocess \
    --frame-channel-name=StartFrameResonant \
    --stim-channel-name=LEDSyncSignal \
    --piezo-period-frames=7 \
    --piezo-skip-frames=3

Command: qa

The qa command makes some QA plots to understand if the stim effects are being correctly removed during preprocessing. It plots a number of frames (given by --max-frames) containing stims, showing the data before and after stim removal.

This is an optional step.

Example:

2p \
    --base-path /my/data \
    --acquisition 20210428M198/slm-001 \
    qa

Command: analyze

The analyze command runs Suite2p on the preprocessed dataset.

Example:

2p \
    --base-path /media/hdd0/two-photon/drinnenb/work \
    --acquisition 20210428M198/slm-001 \
    analyze

Example of analyzing multiple acquisitions together:

2p \
    --base-path /media/hdd0/two-photon/drinnenb/work \
    --acquisition 20210428M198/slm-001 \
    analyze --extra-acquisitions 20210428M198/slm-000

Example of using non-default Suite2p options file (json format):

2p \
    --base-path /media/hdd0/two-photon/drinnenb/work \
    --acquisition 20210428M198/slm-001 \
    analyze --suite2p-params-file two_photon/ops_files/drinnedb.json

Command: backup

The backup command copies the output of one or more stages to backup directory.

2p \
    --base-path /media/hdd0/two-photon/drinnenb/work \
    --acquisition 20210428M198/slm-001 \
    backup \
    --backup-path /media/hdd1/oak/mount/two-photon/backup \
    --backup-stage raw,tiff

--backup_path"

Using multiple commands at once

Several commands can be run in succession by adding each one to your command line with its necessary flags.

2p \
    --base-path /media/hdd0/two-photon/drinnenb/work \
    --acquisition 20210428M198/slm-001 \
    raw2tiff \
    convert --channel 3 \
    preprocess --stim-channel-name=respir \
    analyze --extra-acquisitions 20210428M198/slm-000

Ripping Containers

Ripping is the process for converting a Bruker RAWDATA file into a set of TIFF files.

Containers exist to help run the ripping on any platform, but it has been found they perform sub-optimally and are 10-100x slower than ripping on a Windows machine using the native ripper. It is advised NOT to use this yet.

The lab has created Docker and Singularity containers with the Bruker Prairie View software, which can be used to rip raw data computers with either set of container software installed.

Ripping via Singularity

If you would like to run from a container on Sherlock, the lab keeps a copy available in $OAK/pipeline/bruker-rip/containers.

Here's a quick demo:

$ mkdir -p $OAK/users/${USER}/test
$ cp -r $OAK/pipeline/bruker-rip/sampledata/overview-023 $OAK/users/${USER}/test
$ chmod -R u+w $OAK/users/${USER}/test/overview-023  # Write permissions needed to convert files.
$ cd $OAK/users/${USER}/test/overview-023
$ singularity run --bind=$(pwd):/data $OAK/pipeline/bruker-rip/containers/bruker-rip.sif

Copying wine environment.

Executing rip. One err and four fixme statements are OK.

2020-11-16 17:25:43.859 rip:50 INFO Data created with Prairie version 5.4, using ripper: /apps/Prairie View 5.5/Utilities/Image-Block Ripping Utility.exe
2020-11-16 17:25:43.861 rip:77 INFO Ripping from:
 /data/Cycle00001_Filelist.txt
 /data/CYCLE_000001_RAWDATA_000025
2020-11-16 17:25:43.883 rip:123 INFO Watching for ripper to finish for 3600 more seconds
000d:err:menubuilder:init_xdg error looking up the desktop directory
0031:fixme:ntdll:EtwEventRegister ({5eec90ab-c022-44b2-a5dd-fd716a222a15}, 0x5571000, 0x5582030, 0x5582050) stub.
0031:fixme:ntdll:EtwEventSetInformation (deadbeef, 2, 0x557fd70, 43) stub
0031:fixme:nls:GetThreadPreferredUILanguages 00000038, 0x4fccdb4, 0x4fccdd0 0x4fccdb0
0031:fixme:nls:get_dummy_preferred_ui_language (0x38 0x4fccdb4 0x4fccdd0 0x4fccdb0) returning a dummy value (current locale)
2020-11-16 17:25:53.889 rip:134 INFO   Found filelist files: None
2020-11-16 17:25:53.889 rip:135 INFO   Found rawdata files: None
2020-11-16 17:25:53.889 rip:136 INFO   Found this many tiff files: 1
2020-11-16 17:25:53.889 rip:123 INFO Watching for ripper to finish for 3590 more seconds
2020-11-16 17:26:03.899 rip:134 INFO   Found filelist files: None
2020-11-16 17:26:03.899 rip:135 INFO   Found rawdata files: None
2020-11-16 17:26:03.899 rip:136 INFO   Found this many tiff files: 1
2020-11-16 17:26:03.899 rip:139 INFO Detected ripping is complete
2020-11-16 17:26:13.909 rip:141 INFO Killing ripper
2020-11-16 17:26:13.910 rip:143 INFO Ripper has been killed
2020-11-16 17:26:14.912 rip:115 INFO cleaned up!
X connection to :99 broken (explicit kill or server shutdown).
X connection to :99 broken (explicit kill or server shutdown).

Here's how to run on your own data. We request a node allocation using sdev as long-running jobs should not use login nodes.

$ cd my/data/path
$ sdev  # May take some time to get a machine for development use
$ singularity run --bind=$(pwd):/data $OAK/pipeline/bruker-rip/containers/bruker-rip.sif

[Similar output as above]

And here's how to run a batch job, using the rip.sbatch script from this repository.

$ cd my/data/path
$ sbatch path/to/two-photon/rip.sbatch .
Submitted batch job ABCDEFGH

Ripping via Docker

You can run on a device with Docker installed using the command below. The image will be available locally if you've build from source (see below), or it will be fetched from the the Stanford GitLab. Contact [email protected] if you need access.

$ ./rip_docker.sh \
    scr.svc.stanford.edu/deisseroth-lab/bruker-rip:20200903 \
    /path/to/data/with/filelist/and/rawdata/

Example run:

$ ./rip_docker.sh \
    scr.svc.stanford.edu/deisseroth-lab/bruker-rip:20200903 \
    /media/hdd0/two-photon/sample/overview-023
Setting up wine environment

Executing rip.  It is OK to see 1 err and 4 fixme statements in what follows

2020-09-03 14:41:33.936 rip:50 INFO Ripping from:
 /data/Cycle00001_Filelist.txt
 /data/CYCLE_000001_RAWDATA_000025
2020-09-03 14:41:33.940 rip:96 INFO Waiting for ripper to finish: 3600 seconds remaining
000d:err:menubuilder:init_xdg error looking up the desktop directory
0031:fixme:ntdll:EtwEventRegister ({5eec90ab-c022-44b2-a5dd-fd716a222a15}, 0xd441000, 0xd452030, 0xd452050) stub.
0031:fixme:ntdll:EtwEventSetInformation (deadbeef, 2, 0xd44fd70, 43) stub
0031:fixme:nls:GetThreadPreferredUILanguages 00000038, 0xdaacdb4, 0xdaacdd0 0xdaacdb0
0031:fixme:nls:get_dummy_preferred_ui_language (0x38 0xdaacdb4 0xdaacdd0 0xdaacdb0) returning a dummy value (current locale)
2020-09-03 14:41:43.951 rip:107 INFO   Found filelist files: None
2020-09-03 14:41:43.951 rip:108 INFO   Found rawdata files: None
2020-09-03 14:41:43.951 rip:109 INFO   Found this many tiff files: 1
2020-09-03 14:41:43.951 rip:96 INFO Waiting for ripper to finish: 3590 seconds remaining
2020-09-03 14:41:53.962 rip:107 INFO   Found filelist files: None
2020-09-03 14:41:53.962 rip:108 INFO   Found rawdata files: None
2020-09-03 14:41:53.962 rip:109 INFO   Found this many tiff files: 1
2020-09-03 14:41:53.963 rip:112 INFO Detected ripping is complete
2020-09-03 14:42:03.973 rip:114 INFO Killing ripper
2020-09-03 14:42:03.973 rip:116 INFO Ripper has been killed
2020-09-03 14:42:04.975 rip:88 INFO cleaned up!

Building Containers

To build all available containers, which will first build the Docker container, and then convert it to a Singularity container:

make build

To build just the docker containers:

make build_docker

View the Makefile for additional targets, including targets to build just build specific containers.

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Common scripts, libraries, and utilities for 2p experiments

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