Non-Uniform Depletion Across Targeted Sequences

Symptom

Depletion efficiency varies significantly across different targeted sequences. Some target regions show good depletion while others remain at high levels.

1. 1 Probe amount not optimized for specific sample composition and target abundance
2. 2 Relative probe concentration insufficient for high-abundance target regions
3. 3 Probe pool ratio not adjusted for differential target expression levels
4. 4 Sample-specific factors (secondary structure, accessibility) affect probe binding differently across targets

1. 1 Titrate total amount of probe pool used to optimize for your specific sample type
2. 2 Increase relative probe amount for specific targeted regions showing poor depletion
3. 3 Adjust probe pool composition by spiking in additional probes for under-depleted regions
4. 4 Perform pilot experiments with varying probe:target ratios to identify optimal conditions
5. 5 Consider target RNA secondary structure and redesign probes for problematic regions

Related Video (2)

RocheSequencingUSA ★ 72

The Importance of Performing Ribosomal RNA Depletion

"Explains rRNA depletion methods and their impact on RNA-seq quality, providing context for understanding depletion efficiency issues"

the bumbling biochemist ★ 68

rRNA depletion strategies

"Covers multiple rRNA depletion strategies that could help contextualize why probe optimization varies across different targeted sequences"