Alternative splicing from RNA-seq data

This mini-protocol produces normalized, gene-annotated splicing phenotypes for splicing-QTL analysis using LeafCutter.

Miniprotocol Timing¶

Timing: TBD

Overview¶

The splicing-calling module quantifies splicing with LeafCutter, which derives intron excision ratios from STAR splice-junction files without relying on a transcript annotation.

The normalization module performs missingness and variability filtering followed by quantile normalization. The gene-annotation module then assigns coordinates and phenotype groups required by TensorQTL.

Steps¶

Analysis goal	Commands to run, in order	Inputs
LeafCutter from aligned RNA-seq data	1 → 2 → 3	`output/rnaseq/protocol_example.rnaseq.bam.list.txt`; STAR/WASP alignment directories
LeafCutter from a precomputed ratio matrix	2 → 3	`input/rnaseq/protocol_example.leafcutter.intron_usage_perind.counts.gz`; `tests/fixtures/gene_annotation/protocol_example.leafcutter.intron_count.tsv`

Within the selected route, run the commands in numerical order. The bundled example data support the precomputed-matrix route; the calling route requires aligned BAM files.

1. Quantify intron usage with LeafCutter¶

What it does: leafcutter extracts splice junctions and clusters introns to calculate per-sample intron excision ratios.

sos run pipeline/splicing_calling.ipynb leafcutter   --cwd output/splicing/leafcutter   --samples output/rnaseq/protocol_example.rnaseq.bam.list.txt   --data-dir output/rnaseq/star_output_wasp

2. Normalize LeafCutter ratios¶

What it does: leafcutter_norm filters introns and clusters, mean-imputes retained missing values, and quantile-normalizes the ratio matrix.

sos run pipeline/splicing_normalization.ipynb leafcutter_norm   --cwd output/splicing/leafcutter   --ratios output/leafcutter/normalize/protocol_example.leafcutter.intron_usage_perind.counts.gz   --mean-impute

3. Annotate LeafCutter phenotypes¶

What it does: annotate_leafcutter_isoforms maps introns to genes and writes the coordinate-sorted phenotype matrix and phenotype-group file used by TensorQTL.

sos run pipeline/gene_annotation.ipynb annotate_leafcutter_isoforms   --cwd output/splicing/leafcutter   --phenoFile output/gene_annotation/protocol_example.leafcutter.intron_usage_perind.counts.gz_raw_data.qqnorm.txt   --intron-count tests/fixtures/gene_annotation/protocol_example.leafcutter.intron_count.tsv   --coordinate-annotation tests/fixtures/gene_annotation/Homo_sapiens.GRCh38.103.collapse_only.gene.chr22.gtf.gz   --map-stra site

Output¶

Route/step	Output filename and relative path	Description
LeafCutter calling	`output/splicing/leafcutter/_intron_usage_perind.counts.gz`; `_intron_usage_perind_numers.counts.gz`	Intron excision ratios and supporting intron counts
LeafCutter normalization	`input/rnaseq/protocol_example.leafcutter.intron_usage_perind.counts.gz_raw_data.txt`; `input/rnaseq/protocol_example.leafcutter.intron_usage_perind.counts.gz_raw_data.qqnorm.txt`	QC-filtered and quantile-normalized ratios
LeafCutter annotation	`output/splicing/leafcutter/protocol_example.leafcutter.intron_usage_perind.counts.gz_raw_data.qqnorm.formated.bed.gz`; `output/splicing/leafcutter/protocol_example.leafcutter.intron_usage_perind.counts.gz_raw_data.qqnorm.phenotype_group.txt`	TensorQTL phenotype and grouping files

Anticipated Results¶

Each route ends with a coordinate-sorted, bgzip-compressed phenotype matrix and a matching phenotype-group file. Rows represent introns, columns represent samples, and the normalized values can be supplied directly to splicing-QTL association testing.

Command Interface¶

sos run pipeline/splicing_calling.ipynb -h

sos run pipeline/splicing_normalization.ipynb -h

sos run pipeline/gene_annotation.ipynb -h