The stability of immobilized ASMs through iterations of hybridization, imaging and dehybridization. (A) Generation and decoding of random arrays of ASMs. Padlock probes are mixed with target DNA and (i) correct target recognition enables enzymatic joining of DNA circles. (ii) RCA of the circles generates concatemer products (ASMs) which are (iii) immobilized and labeled with fluorescent tag probes for visualization by fluorescence microscopy. (B) Iterative hybridizations to arrayed ASMs. A series of 10 cycles of hybridizations and dehybridization reactions were carried out to evaluate immobilization chemistry and the efficiency of the hybridization and dehybridization reactions. Two tag probes of the same sequence but labeled with different fluorescence dyes (Cy3 or FITC) were added in alternating cycles of hybridization and dehybridization reactions. The graph illustrates the proportion and fluorescence label of the detected ASMs after each step in the hybridization series. Six image areas were captured per cycle with an average of 7481 counted ASMs. Green label represents labeling with FITC in odd hybridization reactions and red implies labeling with Cy3 in even hybridization reactions. Yellow label represents ASMs that were both green and red, and black ASMs that were not detected in the respective hybridization step. (C) A small subset of raw ASM images illustrates the alternating hybridization and dehybridization cycles. The scale bar is 2 μm.

Multiplex encoding and decoding of genomic loci targeted by selector probes. (A) A genomic sample is prepared by restriction digestion of the sample genome with an appropriate restriction enzyme. (i) Selector probes designed to target specific genomic sequences are added and genomic DNA circles are formed through the action of a thermostable DNA ligase. The circles are either (ii) directly amplified by RCA, or (iii) enriched for by a process which includes restriction digestion of the RCA products into monomers that can be ligated into new circles. The array is created by random immobilization of the ASMs to a poly-l-lysine slide. (B) Arrayed ASMs are decoded by sequential hybridizations of sandwich probes (grey), tag probes (red or blue) and a general tag probe (orange). The sandwich probes contain two regions, one complementary to a specific ASM and one region contains the two decoding tags. These decoding tags, denoted tag 1 and tag 2, are targeted by the tag probes during the hybridization series and their positions in the sandwich probe are illustrated as a pink or blue shade. A small 20 × 20 pixles image subset from one experiment is included, illustrating labeled ASMs after the different hybridization reactions along with an image showing the identified ASMs (scale bar is 2 μm). (C) The decoding scheme used for multiplex decoding of genomic fragments. The names of the gene loci and their corresponding number are listed vertically and the labels from the two tags are illustrated horizontally. Green labels are for Cy3, red labels represent Texas Red and blue correspond to Cy5 labeling. Black means no labeling, i.e. absence of a detectable signal (tag probe).

Evaluation of dynamic range and quantitative precision. Ligated padlock probe A was titrated in steps of 10 in a fixed concentration of ligated padlock probe B (10 pM). The DNA circles were amplified by RCA, arrayed and detected by hybridizing tag probes to A and B specific tags. The counts from A probes ranged from 8 to 19 272 over the dilution series and the average B count was 464. In the graph, the A/B ratios of the two ASM types are plotted on the y-axis, with the corresponding concentrations of ligated padlock probe A on the x-axis. Inset shows a titration with 20% decrements. Axes of the inset are the same as those of the large graph. SDs are from A/B ratios in five imaged areas.

Analysis of relative copy number of loci distributed along chromosomes 13, 18, 21, X and Y. The relative copy-number ratios were obtained by comparing the relative counts of the different ASMs with a normalized reference profile (y-axis). Gain of one copy of an autosomal chromosome should yield a ratio of about 1.5 and a ratio of about 2 would be expected for the X chromosomal loci in female samples. A total loss of a chromosome should yield a value of 0. The ratios are plotted in separate graphs for the different chromosomes. Squares denote male samples and circles female. Filled symbols represent samples from subjects with Down syndrome (trisomy 21) and open symbols represent pooled DNA samples. Median and average ratios from all loci of a chromosome are included to the right in the diagrams.