10x Genomics

, printed on 03/24/2023

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.

To empower our customers with higher sensitivity, Cell Ranger v7.0 includes 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.

Frequently asked questions:

• Why should I include introns for my single cell whole transcriptome gene expression data analysis?
• Should I reanalyze my previous single cell data, if I have not included introns before?
• Are there any biases introduced by using intronic reads?
• I do not want to include intronic reads in my analysis. Can I do that in the upcoming Cell Ranger?
• Can I sequence less if I use intronic mode?
• Can I aggr data run with and without introns?
• Should I include introns for Targeted Gene Expression data analysis?

Example datasets:

• 10k Human PBMCs, 3' v3.1, Chromium X (without intronic reads)
• 10k Human PBMCs, 3' v3.1, Chromium X (with intronic reads)
• 20k Mixture of NSCLC DTCs from 7 donors, 3' v3.1 (without intronic reads)
• 20k Mixture of NSCLC DTCs from 7 donors, 3' v3.1 (with intronic reads)
• 10k Human PBMCs, 5' v2.0, Chromium X (without intronic reads)
• 10k Human PBMCs, 5' v2.0, Chromium X (with intronic reads)
• 10k Bone Marrow Mononuclear Cells (BMMNCs), 5' v2.0 (without intronic reads)
• 10k Bone Marrow Mononuclear Cells (BMMNCs), 5' v2.0 (with intronic reads)

If you have any concerns or questions, please reach out to the 10x Genomics support team at [email protected]