TITLE: Encoding Probe Design using PaintSHOP, SOP006.v1.1
AUTHORS: Nicole Houchins, Rory  Kruithoff, Douglas  Shepherd

[DESCRIPTION]
Document Summary: This document, SOP006 – Encoding Probe Design using PaintSHOP, describes how to  construct RNA-targeting, DNA-based encoding probes with the open-source, interactive application known as  PaintSHOP. Using this website, a desired target refseq ID (DNA or RNA), or gene name, can be input to generate all  of the possible encoding probe target sequences based on your selected criteria and desired stringency. Next,  primers and bridge sequences can be appended to the target sequences. These can be pre-determined and input as  manual files, or you can choose to use literature-established sets using the drop-down menu. Once sequences are  appended, you can download files containing the encoding probe list, order file, reference file and appending file. 


Quick Overview:

Part 1 - Generating encoding probe target sequences 
Step 1 – Determine gene/genes of interest 
Step 2 – Compile your gene RefSeq IDs  
Step 3 - Generate encoding probe sequences using the RNA Probe Design tab 

Part 2 - Appending primers and readout sequences 
Step 1 - Put sequences that will be appended into individual text files 
Step 2 - Append sequences from text files using the Append Sequences tab 

Part 3: Save completed probe files 
Step 1 - Check desired settings for which file will be obtained using the Download tab and save  files.

[GUIDELINES]
v1.1 revision notes 
1. Updated document summary 
2. General update and editing of the document

Adapted from: Hershberg et al, 2021; written by Nicole Houchins and Rory Kruithoff, QI2 Laboratory.


References:  

Hershberg, E. A., Camplisson, C. K., Close, J. L., Attar, S., Chern, R., Liu, Y., ... & Beliveau, B. J. (2021). PaintSHOP enables the  interactive design of transcriptome-and genome-scale oligonucleotide FISH experiments. Nature Methods, 1-8.  https://doi.org/10.1038/s41592-021-01187-3 

Moffitt, J. R., Hao, J., Bambah-Mukku, D., Lu, T., Dulac, C., & Zhuang, X. (2016). High-performance multiplexed fluorescence in situ  hybridization in culture and tissue with matrix imprinting and clearing. Proceedings of the National Academy of Sciences, 113(50),  14456-14461. https://doi.org/10.1073/pnas.1617699113 

[STEPS]
SECTION: Part 1 - Generating encoding probe sequences -  Step 1: Determine gene/genes of interest
1. Determine a gene or list of genes of interest for your specific experimental design. Gene selection may require consideration of expression levels depending on the sample size and distribution of the expression of the  selected genes. For example, a lowly expressed gene (<0.01 FPKM) may be expressed so infrequently that it  may take several hundred cells to see even the occasional FISH spot. The sensitivity of this method does have  limits. Moffitt et al describes FPKM values <0.05 to have substantially reduced correlation with RNAseq data.  To reduce uncertainty, we recommend expression values greater than 1 FPKM and, ideally, values >10 FPKM.
SECTION: Part 1 - Generating encoding probe sequences - Step 2: Compile your gene RefSeq IDs
2. On the UCSC genome browser, https://genome.ucsc.edu/ (or similar), choose the desired species and  assembly.
SECTION: Part 1 - Generating encoding probe sequences - Step 2: Compile your gene RefSeq IDs
3. Enter the gene name of your gene of interest into the Position/Search Term text field. 
SECTION: Part 1 - Generating encoding probe sequences - Step 2: Compile your gene RefSeq IDs
4. Use the RefSeqID from the splice variant that most accurately captures the gene for your purposes.  The Refseq number is formatted as NM_000XXXX and is also known as an accession number. There  may be alternate isoforms of the gene and picking an appropriate isoform accession number is not  always simple. You may need to look closely at the exon coverage to select the most comprehensive  coverage for all isoforms. There are also curated list references and other resources to help with this  selection at https://genome.ucsc.edu/FAQ/FAQgenes.html#whatdo. 
 

SECTION: Part 1 - Generating encoding probe sequences - Step 2: Compile your gene RefSeq IDs
5. Repeat this process for all of the gene targets to compile a list of corresponding RefSeq IDs for your  study.
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
6. Generate your PaintSHOP input files (skip to Part 2 for more information). Because PaintSHOP’s server will  time out in 30 minutes, you may find it more useful to establish files with your appending sequences (Part  2) prior to generating your encoding probe target sequences. This way, your appending files can be quickly  applied to the target sequences in a single session. Below is an example list of the plain text files you should  generate for entering to PaintSHOP.
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
7. Pull up the PaintSHOP website, and open the ‘RNA Probe Design’ tab. 
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
8. Choose the probe set depending on the desired species, then enter all of the RefSeq IDs separated by a  comma and a space (e.g. NM_020309, NM_015267, NM_018008, …) into the “Enter RefSeq IDs  manually” text field. A file can also be uploaded instead of manually entering the RefSeq IDs. In this  text file, the RefSeq IDs should each be placed on their own line and you should then choose ‘custom’  rather than ‘manual’ entry
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
9. Choose the appropriate settings under the Advanced Probe Settings to adjust for off target score and  K-mer count.
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
10. Should you desire the same number of probes per gene of interest, use the ‘Balance Set’ section,  select the number of probes desired and select the ‘balance’ option. 
SECTION: Part 1 - Generating encoding probe sequences - Step 3: Generate encoding probe target sequences using the RNA Probe Design tab
11. Once complete, select ‘submit’ to generate the results. This will provide you a list of the target  sequences as well as a summary for review.  
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 1: Put sequences that will be appended into individual text files
12. For a typical encoding probe set, you will need a set of plain text files (example below), which include  the bridge sequences or primers to be used on either the 5’ or the 3’ end of the encoding probe.  Additionally, you should include a list of the ranges for your bridge sequences as well as a file with a  list of your RefSeq IDs generated from Part 1 above. 
  
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 1: Put sequences that will be appended into individual text files
13. Put each of the sequences (e.g. primers, readout sequences, etc.) that will be appended into text files.  If multiple sequences will be appended for one section of the probe (e.g. multiple readout sequences  for the 5’ and 3’ bridge sequences), separate each sequence by line in the text file. The sequences that go into these files are the exact sequences of the primers or readouts, etc. In PaintSHOP, you will  append the correct sequence for the encoding probes by entering the file as either the forward orientation or reverse complement. To ensure this is easy to double-check, you should include the  input method on the file name as seen in the example above.
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
14. With the Appending files above and Part 1 of this SOP complete, open the Append Sequences tab and  select the type of probe to be used. Choose the RNA Probe Design option under the Design Scheme  section for probes designed to visualize RNA.
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
15. Click ‘Append’ for each of the sections that will have sequences appended to them to reveal more  options. Append the sequence files with the appropriate settings. A few notes on appending:
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
15.1. Note that the 5’ and 3’ Outer Primer are used for our PCR primers, and the 5’ and 3’ Bridge  Sequences are used for readout sequences. 
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
15.2. Within these revealed sections, choose the appropriate settings. These settings include  orientation, format, custom ranges (optional), number per target (optional), select sequence  set, and upload custom set (optional).
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
15.3. Generally, primers will all be the same for all probes while bridge sequences will be ordered  according to the ‘bridge sequence custom ranges’ file.
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
15.4. For the ‘3’ Appending Scheme’ section specifically, select ‘Primer/Bridge/Universal’ option to  reveal more options regarding sections on the 3’ side of the probe. Once selected, you can  choose to append a 3’ primer and bridge sequence.
SECTION: Part 2 - Appending Primers and Readout Sequences - Step 2: Append sequences from text files using the Append Sequences tab
16. Once all of the sequences have been appended, double-check all of the entries and selections and then click ‘Append’ at the bottom of the page.
SECTION: Part 3 - Save completed probe files - Step 1: Check desired settings for which kind of file will be obtained in the Download tab and save files.
17. Open the Download tab.  
SECTION: Part 3 - Save completed probe files - Step 1: Check desired settings for which kind of file will be obtained in the Download tab and save files.
18. Choose the appropriate settings (Appended sequences and RNA Probe Design) regarding the  completed file that will be generated. Such settings include used appending, design scheme, and file  type (order, full probe, appending, or citation). Save the following files.
a. Appending File 
b. Order File 
c. Full Probe File 
d. Reference File (optional)