QC Checkpoint: Purkinje Cell scRNA-seq

Author

Glady Hazitha Samuel

Published

March 17, 2026

Overview

Quality control analysis of raw FASTQ files from GSE256438 (Khouri-Farah et al., 2025) investigating Purkinje cell diversity in embryonic mouse cerebellum.

Dataset: - 3 samples: E16.5 (2 replicates), E18.5 - Technology: 10X Genomics Chromium - Sequencing: Illumina NextSeq 500

Methods

FASTQ files were downloaded from SRA using fasterq-dump and analyzed with FastQC v0.12.1. Results were aggregated using MultiQC v1.15.

Results

General Statistics

Sample Total Reads % Duplicates % GC Status
SRR28065510 (E18.5) 378.4M 82.8% 47% ✅ Pass
SRR28065511 (E16.5 rep2) 415.3M 73.8% 46% ✅ Pass
SRR28065512 (E16.5 rep1) 286.2M 82.9% 47% ✅ Pass

Sequence Quality

Mean Quality Scores

Interpretation: All samples show high base quality (Phred >30) across read positions, with expected slight degradation at read ends.

Duplication Levels

Duplication Levels

Interpretation: High duplication rates (73-83%) are flagged but EXPECTED for scRNA-seq due to: - Low RNA input per cell - PCR amplification during library prep - UMI-based deduplication in Cell Ranger will address this

GC Content

GC Content Distribution

Interpretation: Normal GC distribution (~47%) consistent across samples and appropriate for mouse genome.

Issues Encountered & Solutions

Problem 1: Missing Technical Reads

Issue: Initial download using fasterq-dump --split-files only retrieved Read 2 (cDNA, 98bp). Barcode/UMI reads were discarded.

Evidence: Download logs showed:

reads read: 858,686,424
reads written: 286,228,808  
technical reads: 572,457,616  # <- DISCARDED

Solution: Re-downloading with --include-technical flag to retain all reads required for Cell Ranger.

Problem 2: High Duplication Rates

Issue: FastQC flagged 73-83% duplication as concerning.

Solution: This is normal for scRNA-seq. Cell Ranger’s UMI collapse will remove PCR duplicates during alignment.

Next Steps

  1. Complete re-download with all technical reads (barcodes + UMIs)
  2. Re-run FastQC on complete dataset (R1 + R2)
  3. Cell Ranger alignment to mm10 reference
  4. Seurat QC: filter cells by nFeature, nCount, percent.mt

Session Info

  • FastQC: v0.12.1
  • MultiQC: v1.15
  • Date: March 17, 2026