README.md

Allele Specific Expression (ASE)

This is a ICGC-ARGO pipeline for analysis of allele specific expression (ASE) based on RNA-seq data.

Pipeline

The pipeline processes a reads file (BAM/SAM) and its accompanying variant call files (VCF) together with their matching reference (FA) file and produces, for each SNP, the allele-specific expression ratio and the probability that true ASE is occuring.Using a (GTF) file the positions are also mapped to genes.

If the data are additionally phased, the haplotype-specific expression for each gene is computed.

The whole pipeline operates as illustrated:

The pipeline can be modified using the following quality control parameters:

• QC paramters (applied before ASE read counter)
  • read depth (16)
  • read mapping quality (20)
  • read calling quality (10)
  • read mappability (0.05)

Output

For a file sample_name.bam we obtain the following outputs:

• sample_name.tsv: tab separated document detailing the results of the ASE analysis with the following result columns:
  • ase_ratio: the RAF adjusted for mean bias towards reference
  • ref_bias: the ration of reference counts vs total read counts for the particular base pair
  • AEI_pval: the resulting p-value of binomial statistical test
  • AEI_padj: the p-value corrected using Benjamini/Hochberg false discovery rate correction. The AE is present if p < 0.5.
  • gene_id: if a genome file is provided, maps to an Ensembl gene id
  • gene_feature: if a genome file is provided, maps to an Ensembl gene feature (exon/intron)
• sample_name.gene.log: a log file of the ASE calculation and filtering
• sample_name.vaf.png: a histogram of ase_ratio occurences. In a healthy sample the values should be around 0.5.
• sample_name.hap.tsv: if a genome file is provided and the data are phased, the results of ASE mapped to genes, the result colums are:
  • positions: how many positions are covered by a gene
  • HSE_ratio: ratio of the first haplotype vs. total
  • HEI_pval: the resulting p-value of binomial statistical test for the gene
  • HEI_padj: the FDR B/H p-value correction
• sample_name.hap.log: a log file of the haplotype specific expression calculation

Reference files used

The following human genome files have been tested with the pipeline:

RNA-seq reference genome

https://object.cancercollaboratory.org:9080/swift/v1/genomics-public-data/rna-seq-references/GRCh38_Verily_v1.genome/GRCh38_Verily_v1.genome.fa.gz

mappability file

https://bismap.hoffmanlab.org/raw/hg38/k50.umap.bedgraph.gz

gene mapping

https://object.cancercollaboratory.org:9080/swift/v1/genomics-public-data/rna-seq-references/GRCh38_Verily_v1.annotation/gencode.v40.chr_patch_hapl_scaff.annotation.gtf

Contact

Email questions, feature requests and bug reports to Adam Streck, adam.streck@mdc-berlin.de.

License

icgc-argo-workflows/allele-sepecific-expression is available under the MIT License.

Further reading

Tools and best practices for data processing in allelic expression analysis, Castel et al.