RNA sequencing has transformed the way scientists study gene activity, but many current tools are limited in the kinds of information they can provide. A new pipeline called RnaXtract has been developed to overcome these challenges. Researchers from the Université Laval, Québec designed RnaXtract as an all-in-one solution for bulk RNA sequencing data.

What makes RnaXtract stand out is that it does much more than simply measure gene expression. It also performs variant calling, which identifies changes in the genetic code, and cell-type deconvolution, which helps determine what kinds of cells make up a complex tissue sample. This makes the tool especially valuable for studying diseases that involve many cell types, such as cancer or neurodegenerative disorders.

RnaXtract workflow

Overview of the RNA-seq analysis pipeline, including quality control, pre-processing, variant calling, gene expression quantification, and cell deconvolution. The workflow begins with quality control using FastQC and MultiQC, generating a quality report. Pre-processing includes trimming and preparing FASTQ files. Variant calling involves alignment (STAR), deduplication (Picard), read splitting, and read group addition (GATK), followed by base quality score recalibration (BQSR) and variant identification using HaplotypeCaller and GenotypeGVCFs. Gene expression analysis is performed using Kallisto for quantification and subsequent formatting with the r-rnaseq package, generating gene count and TPM tables. Cell deconvolution is conducted with EcoTyper for ecotype analysis and CIBERSORTx for cell type abundance estimation. Final outputs include tables summarizing gene expression, variant calls, and inferred cell type compositions.

Built on the Snakemake framework, RnaXtract ensures results are reproducible and workflows are efficient. It incorporates cutting-edge tools like EcoTyper and CIBERSORTx to improve accuracy in cell-type analysis. By combining multiple layers of data analysis into one streamlined pipeline, researchers can now gain deeper biological insights into how genes are expressed, how variations in the genome may contribute to disease, and how different cell populations interact within tissues.

RnaXtract represents a significant advancement in the analysis of bulk RNA sequencing, providing scientists with a more complete view of genetic activity and cellular complexity.

Availability – The RnaXtract pipeline is publicly available at https://gitlab-pub.compbio.ulaval.ca/sophiane/rnaxtract