Fluorescent in situ Sequencing (FISSEQ) was originally proposed in 2003. Subsequently, the Church lab and Synthetic Biology Platform of the Wyss Institute developed methods to sequence DNA amplicons on a solid substrate for genome and transcriptome sequencing, resulting in open source DNA sequencing chemistry and hardware platforms. To extend these methods into fixed biological samples, our researchers developed novel sequencing library construction methods.

We developed a method to convert RNA into cDNA in situ within the cells, which is immobilized and amplified locally in a three-dimensional space. The single molecule DNA amplicons are then cross-linked to form a stable matrix, resistant to chemical and thermal perturbation. We have formed these cDNA amplicon matrices in diverse sample types including human iPS cells, fibroblasts, brain and embryo tissue sections, as well as whole embryos. Using a variety of fluorescent sequencing chemistries, including those developed by the Wyss Institute, the sequence of each cDNA amplicon is optically determined, and the expression pattern of each gene is revealed within the natural context of the sample.

This technology represents a high-throughput fluorescent in situ sequencing method (FISSEQ). Our technology allows one to visualize the whole transcriptome with a single nucleotide resolution in individual cells and tissues in situ and identify the functional variant clusters based on the expression pattern. In addition to clustering gene expression patterns based on their spatial organization, our platform enables detection of DNA or RNA based sequence barcodes expressed in multiple cell types under multiple experimental conditions in parallel in situ. FISSEQ further enables the study of the functional organization of tissues and organs, as well as the relationship between genotype and phenotype after genome editing.