Fluorescence in situ hybridization (FISH) allows researchers to visualize the spatial position and quantity of nucleic acids in fixed samples. Recently, considerable progress has been made in developing oligonucleotide (oligo)-based FISH methods that have enabled researchers to study the three-dimensional organization of the genome at super-resolution and visualize the spatial patterns of gene expression for thousands of genes in individual cells. However, there are few existing computational tools to support the bioinformatics workflows necessary to carry out these experiments using oligo FISH probes. Here, we introduce paint server and homology optimization pipeline (PaintSHOP), an interactive platform for the design of oligo FISH experiments. PaintSHOP enables researchers to identify probes for their experimental targets efficiently, to incorporate additional necessary sequences such as primer pairs and to easily generate files documenting library design. PaintSHOP democratizes and standardizes the process of designing complex probe sets for the oligo FISH community.
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The original ‘OligoPaints 2012 hg19’ genome-scale probe collection was downloaded from https://oligopaints.hms.harvard.edu/sites/oligopaints.hms.harvard.edu/files/complete-genome-files/hg19.tar.gz. The original OligoMiner hg19 ‘balance’ genome-scale probe collection was downloaded from https://yin.hms.harvard.edu/oligoMiner/probe_seqs/hg19/hg19b.tar.gz. The original OligoMiner hg38 ‘balance’ genome-scale probe collection was downloaded from https://yin.hms.harvard.edu/oligoMiner/probe_seqs/hg38/hg38b.tar.gz. The original ‘Full 40-mer’ iFISH4u hg19 genome-scale probe collection was downloaded from http://ifish4u.org/custom/dbdownload/hg19.gz. The human hg19 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.fa.gz. The human hg38 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz. The mouse mm9 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/mm9/bigZips/mm9.fa.gz. The mouse mm10 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/mm10.fa.gz. The C. elegans ce11 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/ce11/bigZips/ce11.fa.gz. The D. melanogaster dm6 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/dm6/bigZips/dm6.fa.gz. The zebrafish danRer11 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/danRer11/bigZips/danRer11.fa.gz. The A. thaliana TAIR10 genome assembly was downloaded from https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_chromosome_files/TAIR10_chr_all.fas. The S. cerevisiae sacCer3 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/bigZips/sacCer3.fa.gz. The rat rn6 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/rn6/bigZips/rn6.fa.gz. The chicken galGal5 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/galGal5/bigZips/galGal5.fa.gz. The chicken galGal6 genome assembly was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/galGal6/bigZips/galGal6.fa.gz. All genome-scale probe collections, primer sequences, bridge sequences, SABER-associated sequences, and transcriptome intersects hosted on paintshop.io are available to download from https://github.com/beliveau-lab/PaintSHOP_resources repository. All repositories are available under the MIT license. Raw and processed microscopy images are available upon request.
The source code for the PaintSHOP web application is available as Supplementary Software 1 and at https://github.com/beliveau-lab/PaintSHOP. The source code for the homology optimization pipeline is available as Supplementary Software 2 and at https://github.com/beliveau-lab/PaintSHOP_pipeline.
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We thank G. Nir, D. Shechner, A. Tsue, H. Nguyen, J. Y. Kishi and J. Harke for helpful feedback during the beta testing phase of PaintSHOP, N. Peters and D. Fong for assistance with microscopy, the Genome Sciences IT team for technical assistance and members of the Beliveau laboratory for feedback on the written paper. We also thank S. Lapan and E. West for productive discussions that inspired aspects of this work. This work was supported by a Damon Runyon Dale F. Frey Breakthrough Award (to B.J.B.), the National Institutes of Health (under grant no. 1R35GM137916 to B.J.B.), and the DiaCOMP consortium (under grant no. 19AU3987 to S. Akilesh and B.J.B.). Imaging on the University of Washington W.M. Keck Center Lecia SP8X confocal microscopy was enabled by funding from the NIH (grant no. S10 OD016240). The Nikon CSU-W1 SoRa superresolution microscope was funded by the Howard Hughes Medical Institute. We also thank the Allen Institute for Brain Science founder, P. G. Allen, for his vision, encouragement and support.
The authors declare no competing interests.
Peer review information Nature Methods thanks Zhe Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Hershberg, E.A., Camplisson, C.K., Close, J.L. et al. PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments. Nat Methods 18, 937–944 (2021). https://doi.org/10.1038/s41592-021-01187-3