Guidance Needed: Best Practices for Handling Technical Replicates in RNA-seq Analysis
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@46b41c1d
Last seen 11 hours ago
India

Hello Bioinformatics Community,

I'm currently analyzing an RNA-seq dataset involving subtypes of disease from 16 brain tissue samples, with 2 runs each making 32 SRR runs. Each biological sample has multiple sequencing runs, one sample has two runs, resulting in technical replicates. I'm seeking guidance on the optimal strategy to incorporate these replicates into my differential expression analysis.

Specific Questions:

Merging Technical Replicates:Should technical replicates (multiple sequencing runs from the same biological sample) be merged:

before alignment,

after alignment but before counting, or

after obtaining gene expression counts?

By merging, I mean should I add gene counts?

Downstream Analysis (DESeq2/edgeR):What is the recommended method for handling these technical replicates to ensure accurate and robust differential expression results? Should I use functions such as collapseReplicates (DESeq2) or sumTechReps (edgeR)?

Any recommendations, protocols, or references would be greatly appreciated.

Thank you!

DESeq2 TechnicalReplicates RNASeq • 172 views
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Why was my comment removed? It's irritating that it was moderated away without leaving any note.

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@james-w-macdonald-5106
Last seen 5 hours ago
United States

Please don't cross-post. You have already asked this on biostars.org, which is the correct venue for general questions like this.

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