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NGS Library Preparation (NEBNext Ultra II) moderate

Excessive Adaptor Dimer Formation

Symptom
Sharp 127 bp peak visible on Bioanalyzer representing adaptor dimers, reducing the proportion of desired library fragments and overall usable yield.
Common Causes
  1. 1 Adaptor concentration too high relative to sample input amount, quality or type
  2. 2 Adaptor added directly to ligation master mix instead of to sample first, causing increased self-ligation
  3. 3 Adaptor dilution or titration not optimized when sample source changes (extraction method, tissue type, etc.)
Solutions
  1. 1 To recover samples with adaptor dimers, repeat bead cleanup using 0.9X bead ratio
  2. 2 Optimize adaptor dilution based on sample input, quality and type using adaptor titration experiment
  3. 3 Do NOT add adaptor to ligation master mix; add adaptor to sample first, mix, then add ligase master mix and ligation enhancer
  4. 4 Repeat adaptor titration if sample source changes (e.g., extraction method, tissue type)
Related Video (3)
New England Biolabs ★ 95
NEBNext Ultra II DNA Library Prep Protocol
"This is the exact NEBNext Ultra II DNA Library Prep protocol video, so it directly shows correct technique, reagent handling, and where adaptor dimer artifacts can arise."
New England Biolabs ★ 85
12 Quick Tips for NGS Library Preparation
"Provides practical NGS library prep tips that would help a researcher diagnose and avoid adaptor dimer formation, such as adaptor-to-input ratio guidance."
Illumina ★ 70
How can I tell if I sequenced through the insert? Part 1 | Illumina Video
"Uses Bioanalyzer traces to interpret library insert size and sequencing-through-insert issues, which contextualizes interpreting a 127 bp adaptor dimer peak."
Source: neb.com ↗
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