Why do I have high mitochondrial reads or duplicates in my ATAC-seq data? How can I troubleshoot?

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High mitochondrial reads

  • High-quality ATAC-seq libraries are expected to contain <15% mitochondria reads.

  • Mitochondrial read fraction can be influenced by sample type.

  • Suboptimal sample preparation can increase mitochondrial carryover. See this article for sample-prep guidelines for ATAC-seq.

  • Insufficient removal of supernatant post nuclei extraction contributes to increased mitochondrial signal. Make sure to keep the nuclei pellets uninterrupted and remove as much supernatant as possible after centrifugation (Step 17 in Manual).

High duplicate rate

  • High-quality ATAC-seq libraries are expected to contain <25% duplicate reads.

  • Low nuclei input, low sample-type complexity, and increased PCR amplification cycles (if performed for low-input samples) can reduce library complexity and increase duplication.

  • See this article for guidelines on low input sample optimizations.