Our lab’s research is centered around technologies involving isoform research in mouse and human tissues. Thus, we devised the first long-read approach for thousands of single cells (Single-cell isoform RNA sequencing, ScISOr-Seq, Gupta, …, Tilgner, Nat Biotechnol’18). We then devised novel statistical isoform- testing and the first spatially defined long-read sequencing approach (Slide-isoform sequencing, Sl-ISO-Seq, Joglekar, …, Tilgner, Nat Comms’21 – funded by NIGMS). We complemented this R package with a visualization tool for single-cell isoform expression (ScISOr-Wiz, Stein, …, Tilgner, Bioinformatics’22 – funded by NIGMS). Most recently, we are applying ScISOr-Seq across five adult mouse brain regions and three developmental time points to define the developmental timelines leading to brain-region specific splicing (Joglekar, …, Tilgner, to be submitted to Nature by March – in smaller parts funded by NIGMS). We devised methods to remove intronic cDNAs and artifactual non-barcoded cDNAs introduced by 10xGenomics, enabling single-cell splicing research in frozen tissues (Single-nuclei isoform RNA sequencing, SnISOr-Seq, Hardwick, …, Tilgner, Nat Biotechnol’22 – funded by NIGMS). We recently enhanced our SnISOr-Seq to define dysregulation of AS in neurons and glia from frontotemporal dementia (FTD) cases. We show that the strongest FTD-associated splicing events occur in restricted cell subtypes and that ~20% of these events detected in a cell type cannot be observed in all cells jointly (Belchikov, …, Tilgner, in review – funded by NIGMS). Separately, we defined error sources in long-read data. Specifically, we used barcoding technologies to sequence two cDNA representations of the same RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We found highly specific error patterns (Mikheenko, …, Tilgner, Genome Research’22 – funded by NIGMS). Using the knowledge on such error patterns, we then created accurate isoform detection software (Prjibelski, …, Tilgner, Nat Biotechnol’23). We have described the field in reviews on long-read sequencing (Hardwick, …, Tilgner, Frontiers Genetics’19) and single-cell isoform research (Joglekar, …, Tilgner, in review – funded by NIGMS). Upon invitation by Nature Methods, in honor of their naming of long-read sequencing as the method of the year 2022, we detailed advances and future directions (Foord, …, Tilgner, Nature Methods, in press – funded by NIGMS). Over the next 5 years, my lab will advance what is measurable in single-nuclei and spatial experiments. For example, we will define TSS at single-nucleotide, single-cell and single-molecule resolution using long reads. Moreover, we will measure DNA methylation, open chromatin, gene expression and splicing in individual cells to decipher how these layers influence each other in health and disease. More generally, we will advance what can be measured in single-cell and spatial biology, including but not limited to spliceosomal RNAs, miRNAs and protein-protein interactions that are governed by alternative splicing.

DNA methylation, chromatin, transcription initiation and alternative splicing form a tightly linked triangle and single-cell genomics has revolutionized modern-day biology. However, to-date we are lacking a precise understanding of the relationships between the above modalities in the cell types that form complex tissue. Here, we devise new single-cell technology to measure multiple modalities to infer the relationships between them in health and disease.