We are interested in cell-type specific isoform expression in the brain, however, studies of splicing patterns are difficult to conduct in human because human brain tissue is often frozen, and isolating spliced RNA from frozen tissue is prohibitive on multiple levels. Using a combination of microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed Single-nuclei Isoform RNA sequencing (SnISOr-Seq) and applied it to the analysis of human adult frontal cortex samples. This is all original code generated for processing of data described in our manuscript.
Most of our processing workflow has already been published but this repository contains code for initial data processing with scripts for:
- Extending barcode detection to allow mismatches
- Error correction to prevent 'molecule inflation' in UMI mismatches
- Coordination analysis for exon pairs
- Coordination analysis for exon-endsites
The structure is as follows:
lr_scripts
├── bashScripts
│ ├── parallelizeMolInflation.sh
│ ├── run_11merMatching.sh
│ └── v2.0a_bc_finder.awk
├── pythonScripts
│ ├── barcodeUMI_finder_11mer.py
│ ├── minEditDistance_post11merMatching.py
│ └── moleculeInflation_10x_forParallel.py
└── rScripts
├── endSite_exon_chiSq_countMatrix.R
├── endSite_exon_countMatrix_Generate.R
├── exon_coord_chiSq_countMatrix.R
└── get10xMoleculeInfo.R
In addition, to ensure maximal reproducibility, we have provided the input as well as code for producing (most) panels of the main figures in our text. Code to generate isoform plots can be found in the form of an R-package to be released soon
plotGeneration
├── figure2.R
├── figure3.R
├── figure4.R
├── figure5.R
└── figure6.R
If you want to explore this dataset and query expression of disease-associated exons in our human frontal cortex samples, we have made an interactive portal which allows you to upload lists of exons and see their cell-type specific expression. We have uploaded our lists of ASD, ALS, and SZ- associated exons under the data tab. Feel free to use that as a demo in the online portal. To access the portal, visit isoformAtlas.com --> Access Data --> Exons to view.
We encourage you to email with questions about the data or approach