Instructions for probe design

Caution

You may want to add python and pip as aliases for python3 and pip3

On your terminal;

echo "alias python='python3'" >> ~/.bashrc
echo "alias pip='pip3'" >> ~/.bashrc
source ~/.bashrc

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;

git clone http://github.com/ggirelli/oligo-melting ~/oligo-melting
cd ~/oligo-melting
pip install .

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

mkdir <project_name>
cd <project_name>

Probe design pipeline:

Alternative 1: Normally repetitive regions.

1. Preparation

• The probe desin pipeline data is currently intended to be run on a folder called data/ contained within the pipeline project folder <project_name>.

• 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.

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

• Place your reference genome in the data/ref/ folder. Make sure that the chromosome naming matches with the reference genome name provided in all_regions.tsv.

• The reference folder can alternatively be gathered using prb get_ref_genome. In that case, adjust the script manually with the correct Ensembl address for your genome of interest.

2. Generate all required subfolders inside your project directory:

prb makedirs

3. 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.

4. 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

• 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

Tip

ADD -g if this is the first time running with a new reference genome!

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

prb unfinished_HUSH

5. Recapitulate nHUSH results as a score

prb reform_hush_combined DNA|RNA|-RNA length sublength until

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

6. 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))   

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

prb generate_blacklist -L 40 -c 100

Tip

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

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

8. 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

9. Query the database to get candidate probes:

prb cycling_query -s DNA -L 40 -m 8 -c 100 -t 40 -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.

10. Summarize the final probes:

prb summarize_probes_final

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

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.

Caution

This approach occupies a lot more hard drive space!

1. 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.

2. (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.

2. Generate all required subfolders:

prb makedirs

3. 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

Note

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

4. Apply the region exclusion mask on the reference genome.

prb exclude_region

5. 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

6. 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

Note the _excl specific to the exclusion mode.

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

prb unfinished_HUSH

7. 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).

8. 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))

9. 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

10. 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.

11. 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.