Genome Annotation Version Mismatch Between Design and Analysis

Symptom

Target sequence used to design probes differs from the reference used to evaluate depletion. Depletion appears ineffective when analyzed against current genome annotations.

1. 1 Outdated transcriptome/genome version used to extract target RNA sequence for probe design
2. 2 Different genome assembly versions between probe design and sequencing analysis pipelines
3. 3 Genome annotations evolved between probe design and evaluation timepoints
4. 4 Inconsistent reference databases used across experimental workflow

1. 1 Check for differences between transcriptome/genome versions used for probe design vs depletion evaluation
2. 2 Use identical genome assembly and annotation versions throughout entire workflow
3. 3 Document genome version (e.g., GRCh38.p13, mm10) used at each step
4. 4 Re-design probes using current genome annotations if significant version differences exist
5. 5 Maintain version-controlled reference genome files for reproducibility

Related Video (2)

Bilibili (China-Accessible Mirrors) ★ 72

LNA Technology for NGS and PCR Optimization

"Demonstrates LNA-based probe design and rRNA depletion methodology, directly relevant to understanding how probe-target matching affects depletion efficacy"

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The Importance of Performing Ribosomal RNA Depletion

"Explains rRNA depletion methods and their impact on RNA-seq results, providing experimental context for why depletion failures occur and affect downstream analysis"