Additional high molecular weight peak at ~1,000 bp

Symptom

Bioanalyzer shows an additional peak at higher molecular weight than expected library size (approximately 1,000 bp). This represents single-stranded library products that have self-annealed to form heteroduplexes.

1. 1 PCR over-amplification with excessive cycle number
2. 2 PCR input amount was too high for the reaction
3. 3 Primers become limiting in late PCR cycles
4. 4 Adaptor sequences on fragment ends anneal to each other instead of primers
5. 5 Formation of heteroduplexes with different insert sequences that migrate slower

1. 1 Reduce number of PCR cycles according to input amount
2. 2 Optimize PCR input amount to prevent late-cycle artifacts
3. 3 Perform titration experiment to determine optimal cycle number
4. 4 Note: If ratio is low compared to library, may not significantly impact sequencing
5. 5 Monitor amplification in real-time if using qPCR to stop at exponential phase

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