probe-design

Instructions for probe design

[!CAUTION] You may want to add pip as alias for pip3

On your terminal;

echo "alias pip='pip3'" >> ~/.bashrc
source ~/.bashrc

Or simply use pip3 instead of pip

Installation

• Install probe_design (also installs ifpd2q)

pip install probe_design -U

This adds prb (short for probe design) as a shell command.

• Install oligo-melting

On your terminal;

pip install git+https://github.com/ggirelli/oligo-melting.git

[!NOTE] nHUSH, HUSH and escafish are private repositories

• Install the dev branch of nHUSH

• Install HUSH

• Install escafish

• Install OligoArrayAux

Preparation

DNA:

• Get the genomic coordinates of the regions of interest

• Get the reference genome

RNA:

• Get the transcripts of interest

• Get the reference transcriptome

Notes:

• For DNA probes, the reference genome will be used both to extract the sequences of interest and to test probe candidates for homology. If different genomes need to be used, follow RNA steps and provide the regions of interest directly.

• For combined DNA-RNA FISH, the probe sets should be designed with an homology check against both genome and transcriptome.

• All the commands below assume you are starting from your project directory

• To make a project directory and change directory to project directory

mkdir <project_name>
cd <project_name>

Probe design pipeline:

Alternative 1: Normally repetitive regions.

• Preparation

Inside the project dirctory.

prb makedirs

This will create data directory and its subdirectories data/rois and data/ref.

• Upon starting the pipeline, the data/ folder should only contain data/rois/ and data/ref/ (and possibly data/blacklist/, see 6.). If more folders are included, consider making a back-up or simply removing them.

• Input file for Region of Intrests (ROIS)

[!CAUTION]

• your region of interests file MUST be named all_regions.tsv
• all_regions.tsv MUST follow the EXAMPLE format.
• all_regions.tsv MUST be placed within data/rois folder.

• List your regions of interest and their coordinates in the input file: data/rois/all_regions.tsv

• Download Reference genome

For CHM13 T2T

prb get_T2T

options

-p: prefix for the chromosomes ;default: CHM13.T2T names will prefix.chromosome.ID.fa where ID stands for chromosome ID i.e., 1-22+X,Y,M

For GRCh38

prb get_GRC -split

usage: prb get_GRC [-h] [-s {homo_sapiens,mus_musculus}] [-b BUILD] [-r RELEASE] [-d DIR] [-f FILENAME] [-k] [-split]

download ensemble genome

options:
-h, --help
------> show this help message and exit
-s {homo_sapiens,mus_musculus}, --species {homo_sapiens,mus_musculus}
-b BUILD, --build BUILD
------> the build number of the genome
-r RELEASE, --release RELEASE
------> release number of the build
-d DIR, --dir DIR destination directory
-f FILENAME, --filename FILENAME
------> give a specific name to the downloaded file
-k, --keep
------> whether to keep gzip files
-split
------> whether to split into chromosomes

• Retrieve your region sequences and extract all k-mers of correct length:

prb get_oligos DNA|RNA [optional: applyGCfilter 0|1]
# Example:
prb get_oligos DNA 1

[!NOTE] If indicating RNA, the module will assume that the transcript / region sequences are already present in the data/regions folder. Default: `DNA.

• Test all k-mers for their homology to other regions in the genome, using nHUSH. Instead of running the entire k-mers (of length L) at once, can be sped up by testing shorter sublength oligos (of length l). -m number of mismatches to test for (always use 1 when running sublength); -t number of threads, -i comb size

[!CAUTION] Make sure your Length (-L) here matches with the Length in your all_regions.tsv file

• Full length:

prb run_nHUSH -d RNA -L 35 -m 5 -t 40 -i 14

• Sublength:

prb run_nHUSH -d DNA -L 40 -l 21 -m 3 -t 40 -i 14

prb run_nHUSH -d {DNA|RNA} -L {length} -l (optional){sublength} -m {number of mismatches} -t {threads} i {comb size}

• In case nHUSH is interrupted before completion, run before continuing:

prb unfinished_HUSH

• Recapitulate nHUSH results as a score

prb reform_hush_combined DNA|RNA|-RNA length sublength until

e.g., prb reform_hush_combined DNA 40 21 3

(until denotes the same number as specified after -m when running nHUSH).

• Calculate the melting temperature of k-mers and the free energy of secondary structure formation:

prb melt_secs_parallel (optional DNA(ref) | RNA(rev. compl))   

e.g., prb melt_secs_parallel DNA

• Generate a black list of abundantly repeated oligos in the reference genome.

[!NOTE] This only needs to be run once per reference genome if not using any exclusion regions! Just save the blacklist folder between runs.

prb generate_blacklist -L 40 -c 100

L: oligo length
c: min abundance to be included in oligo black list

• Create k-mer database, convert to TSV for querying and attribute score to each oligo (based on nHUSH score, GC content, melting temperature, homopolymer stretches, secondary structures).

prb build-db_BL -f q_bl -m 32 -i 6 -L 40 -c 100 -d 8 -T 72

m: Maximum length of a consecutive match. Default: 24
i: Maximum length of a consecutive homopolymer. Default: 6
All oligos with a longer consecutive match or homopolymer are stricly excluded.
L: oligo length
c: min number of occurrences for an oligo to be counted in black list
(should match settings used in 6.)
d: min Hamming distance to an oligo in the blacklist for exclusion
T: Target melting temperature. Default: 72C

• Query the database to get candidate probes:

prb cycling_query -s DNA -L 40 -m 8 -c 100 -t 40 -g 500 -greedy

[optional: -greedy. Speed > quality] [optional: -start 20 -end 100 -step 5]

To sweep different oligo numbers, otherwise uses the oligo counts provided in ./rois/all_regions.tsv [optional: -stepdown 10] Number of oligos to decrease probe size with every iteration that does not find enough oligos. Default: 1

Cycling query which generate probe candidates, then checks the resulting oligos using HUSH, removes inacceptable oligos and generate probes again. If enough oligos cannot be found, design probes with fewer oligos, decreasing with stepdown at each step.

• Summarize the final probes:

prb summarize_probes_final

Some visual elements can be obtained using the following notebooks (TODO!):

prb plot_probe_candidates
prb plot_oligos

Alternative 2: Repetitive or repeated regions.

In this alternative, the region (along with any user-indicated repeats) is masked out from the reference genome used by nHUSH. This way, repeated oligos that are specific for the ROI can be included in the final probe.

Warning: This approach occupies a lot more hard drive space!

• Generate all required subfolders:

prb makedirs

• Input file for Region of Intrests (ROIS)

[!CAUTION]

• your region of interests file MUST be named all_regions.tsv
• all_regions.tsv MUST follow the EXAMPLE format.
• all_regions.tsv MUST be placed within data/rois folder.

• Additional Preparation

• Besides data/rois/ and data/ref/, the pipeline requires an additional data/exclude/ folder containing BED files with the coordinates of sections to mask out when running HUSH for each ROI.

• Download Reference genome

For CHM13 T2T (advised for repetetive regions)

prb get_T2T

options

-p: prefix for the chromosomes ;default: CHM13.T2T names will prefix.chromosome.ID.fa where ID stands for chromosome ID i.e., 1-22+X,Y,M

• (UNLESS manually providing exclusion regions) Exclude regions of interest from HUSH scan.

prb generate_exclude

• The same sheet template can be used to manually add further regions to exclude.

• Retrieve your region sequences and extract all k-mers of correct length:

# (from Pipeline/)
prb get_oligos DNA|RNA [optional: applyGCfilter 0|1]
# Example:
prb get_oligos DNA

If indicating RNA, the module will assume that the transcript / region sequences are already present in the data/regions folder. Default: `DNA.

• Apply the region exclusion mask on the reference genome.

prb exclude_region

• Generate a black list of abundantly repeated oligos in the reference genome.

prb generate_blacklist -L 40 -c 100

Needs to be re-run everytime when using exclusion masks. L: oligo length; c: min abundance to be included in oligo black list

• Test all k-mers for their homology to other regions in the genome, using nHUSH. Instead of running the entire k-mers (of length L) at once, can be sped up by testing shorter sublength oligos (of length l). -m number of mismatches to test for (minimum 1 for sublength; more gives better information but takes longer time); -t number of threads, -i comb size

Sublength:

prb run_nHUSH_excl -d DNA -L 40 -l 21 -m 3 -t 40 -i 14

prb run_nHUSH_excl -d {DNA|RNA} -L {length} -l (optional){sublength} -m {number of mismatches} -t {threads} i {comb size}

Note the _excl specific to the exclusion mode.

In case nHUSH is interrupted before completion, run before continuing:

prb unfinished_HUSH

• Recapitulate nHUSH results as a score

# Format:
prb reform_hush_combined DNA|RNA|-RNA length sublength until
# Example:
prb reform_hush_combined DNA 40 21 3

(until denotes the same number as specified after -m when running nHUSH).

• Calculate the melting temperature of k-mers and the free energy of secondary structure formation:

prb melt_secs_parallel (optional DNA(ref) / RNA(rev. compl))

e.g., prb melt_secs_parallel DNA

• Create k-mer database, convert to TSV for querying and attribute score to each oligo (based on nHUSH score, GC content, melting temperature, homopolymer stretches, secondary structures).

Recommended:

prb build-db_BL -f q_bl -m 32 -i 6 -L 40 -c 100 -d 8 -T 72

f: score function
d: max Hamming distance to blacklist that is excluded
L: oligo length
c: min abundance to be included in oligo blacklist
i: max identical consecutive base pairs,
T: target temperature
m: max length of consecutive off-target match

• Query the database to get candidate probes:

prb cycling_query -s DNA -L 40 -m 8 -c 100 -t 40 -g 500 -stepdown 50 -greedy -excl

[optional: -greedy. Speed > quality] [optional: -start 20 -end 100 -step 5]

To sweep different oligo numbers, otherwise uses the oligo counts provided in ./rois/all_regions.tsv [optional: -stepdown 10] Number of oligos to decrease probe size with every iteration that does not find enough oligos. Default: 1

Cycling query which generate probe candidates, then checks the resulting oligos using HUSH, removes inacceptable oligos and generate probes again. If enough oligos cannot be found, design probes with fewer oligos, decreasing with stepdown at each step.

• Summarize the final probes:

prb summarize_probes_final

Generate probes for ordering

• Select forward, reverse primers and color flaps.
• Add the forward and reverse primer sequences to the probe oligos
• The forward primer to order has the color flap + the forward sequence
• The reverse primer to order has the t7 promoter sequence + the rev. compl of the rev sequence in the oligo
• The complete oligos can be uploaded as an Excel file containing the oligo names (arbitrary but unique) and the sequences

TO DO:

• Adapt the code for more flexibility in input/output folders.
• Add a visual report of the probes at the end of the pipeline.
• One-button process!
• Find a way to automatize selecting primer sequences.