Thank you, I followed the GATK pipeline.
Hi All,
I want to perform the gene centered allele specific expression (ASE) analysis. How to get the read counts for reference allele and alternate allele from aligned bam file (RNA-seq data). I have found several tools for ASE once we have counts data.
Can you provide some tools to get the counts data or direction to proceed.
Thank you.
3 answers
The first step is to idetifying polymorphisms of your libraries against the reference genome. I would go for a pseudo-reference aproach which implies that you impute the homozygous SNPs of your libraries into the reference genome. Then you align your libraries against both the reference and the pseudo-reference to retrieve reciprocal calls that are heterozygous. I would go for the GATK's pipeline, if you follow this pipeline you will finish with a VCF file that contains the number of reads that mapped to the reference/alternative alleles. Then you normalize and perform differential expression analysis (binomial test/ LRT) to detect allelic imbalance at heterozygous SNPs. This was the pipeline I developed to analyze transcriptomic data for ASE analysis.
Best
You may want to try Allis. It requires an RNA-Seq BAM and optionally variant calls from WGS or WES. If no variants are provided they are called from the RNA-Seq BAM file. The variants are then phased against the 1000 Genomes reference panel (hg19) using Eagle2. Allis then parses the phased VCF file for het. bi-allelic variants which are used to split the input BAM into haplotype 1 and haplotype 2, ambiguous reads are discarded. In addition to h1.bam and h2.bam it outputs an allele-specific count table with depth, REF-support, ALT-support, the variant allele frequency and the haplotype allele frequency. It also runs a simple binomial test for every SNP. It's still work-in-progress so any feedback is welcome.
Thank you, I followed the GATK pipeline. I will take a look at the Allis as well.
GATK tool has the option of running without phasing information.
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How did you map your reads? Did you construct personalized haplotype genomes? Or did you align to a single reference?
I aligned to single reference genome hg19.