Adaptor-dimer peak at approximately 127 bp

Symptom

Bioanalyzer shows a distinct peak at approximately 127 bp representing adaptor-dimer formation. These dimers will cluster and be sequenced, potentially consuming sequencing capacity.

1. 1 Addition of non-diluted adaptor during ligation setup
2. 2 RNA input amount was too low for the reaction
3. 3 RNA was over-fragmented during fragmentation step
4. 4 RNA loss occurred during fragmentation procedure
5. 5 Inefficient ligation reaction conditions

1. 1 Dilute adaptor before setting up ligation reaction according to protocol
2. 2 Increase RNA input amount to recommended range
3. 3 Optimize fragmentation time to prevent over-fragmentation
4. 4 Clean up PCR reaction again with 0.9X SPRIselect Beads or NEBNext Sample Purification Beads
5. 5 Note: If adaptor-dimer ratio is low compared to library, sequencing may proceed but some reads will be dimers

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