PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments

Abstract

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.

Fig. 1 |. The PaintSHOP workflow.

a, Overview of the genome-scale FISH probe collection design process. b, Overview the probe set creation functionality of the PaintSHOP web application.

Fig. 2 |. Probe counts for each human probe collection included in PaintSHOP.

a, Counts of the number of candidate probes before any downstream processing was performed. b, The number of candidates after filtering for probes with greater than 100 off-target alignments. c, The number of remaining probes with no off-target alignments. d, The number of remaining probes with between 1 and 100 off-target alignments.

Fig. 3 |. Transcriptome and genome coverage of probe collections.

a, The number of probes per RefSeq transcript annotation (”all”; green, left side of violins) or isoform-flattened RefSeq gene annotation (”iso-flat”; blue, right side of violins) in the genome-scale probe collections hosted by PaintSHOP. Annotations with >250 probes were plotted with a value of 250 to simplify presentation. b, The number of probes per 10-kilobase window in the genome-scale probe collections hosted by PaintSHOP. Each genome was split into adjacent (i.e. non-overlapping) windows. Violin plots in a, b show a kernel density estimation (blue, green) along with traditional boxplot elements: median—white dot, quartiles 2 and 3—thick black box, quartile 1 value − 1.5 * (quartile 3 value – quartile 1 value)—lower bound of black line, quartile 3 value + 1.5 * (quartile 3 value – quartile 1 value)—upper bound of black line. The minimum value of the kernel density estimation was set to 0 for display purposes. In a, annotations with >250 probes mapping to them had their value set to ‘250’ for display purposes.

Fig. 4 |. Multiplexed DNA FISH programmed by PaintSHOP.

a, Overview of 30-target human X chromosome library design. Each site is individually addressable. b, A three-color “side-by-side” pattern imaged on 46 XX human metaphase chromosome spreads. c, A three-color “repeat” pattern imaged on 46 XX human metaphase chromosome spreads. Scale bars, 5 µm (spreads) or 20 µm (fields of view). Each pattern was visualized in 3 independent samples and yielded similar results.