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Recommendation on Including Introns for Gene Expression Analysis

Recommendation on Including Introns for Gene Expression Analysis

Updated: July 22, 2022

In 10x Genomics whole transcriptome Gene Expression data, intronic mapped reads account for 20-40% of the reads. These reads were not counted by default in past versions of Cell Ranger. Recent data has indicated that intronic reads are usable data (they arise from poly-A tracts in the transcripts, and are not generated via priming from genomic DNA). Including intronic reads, for both cellular and nuclei samples, could lead to higher sensitivity (higher UMI counts, more genes per cell) and less wasted sequencing.

This Technical Note demonstrates how including intronic reads in Cell Ranger analysis resulted in more usable data and higher sensitivity for Single Cell 3’ and 5’ Gene Expression cellular datasets. In addition, for a Single Cell 3' Gene Expression 10k peripheral blood mononuclear cell (PBMC) dataset, cell type annotation and differential gene expression analyses were comparable with or without intronic reads.

New in Cell Ranger v7.0: Intronic reads are counted by default for whole transcriptome gene expression data.

To empower our customers with higher sensitivity, Cell Ranger v7.0 and later include intronic reads by default towards UMI counting. Intronic reads can be included manually in your analysis with Cell Ranger v5.0 - 6.1 with the following options:

  • If you are running the cellranger count pipeline, you can add the --include-introns flag to the command.
  • If you are running the cellranger multi pipeline for 3' Single Cell Gene Expression products or 5' Single Cell Immune Profiling products, you can add include-introns,true to the [gene-expression] section of Multi Config CSV.

If you have any concerns or questions, please reach out to the 10x Genomics support team at support@10xgenomics.com.