Home Genetics / Genomics 12 Quick Tips for NGS Library Preparation
Steps
  1. 1 Prepare vials and minimize contamination --:--
  2. 2 Maintain aseptic technique for RNA work 00:17
  3. 3 Store samples and reagents properly 00:37
  4. 4 Handle and preserve isolated RNA 00:50
  5. 5 Mix reaction components efficiently 01:05
  6. 6 Add reagents and manage enzymes 01:23
  7. 7 Store final library samples correctly 01:37
Genetics / Genomics New England Biolabs

12 Quick Tips for NGS Library Preparation

Protocol
Difficulty
intermediate

Steps

1
Prepare vials and minimize contamination

Spin down all vials before opening to ensure contents settle at the bottom. Use filter pipette tips throughout the procedure to minimize contamination during handling.

▶ --:--
2
Maintain aseptic technique for RNA work

Use proper aseptic techniques when working with RNA samples. Clean working surfaces with RNAzap or similar products and change gloves frequently to prevent bacterial and mold contamination from hands and dust particles.

▶ 00:17
3
Store samples and reagents properly

Keep all sample and reagent vials capped when not in use. Use RNA and DNA-free plastics, water, and solutions for all work.

▶ 00:37
4
Handle and preserve isolated RNA

Keep purified RNA on ice at all times and avoid freeze-thaw cycles. Store purified RNA long-term at minus 20 degrees or minus 70 degrees in nuclease-free water.

▶ 00:50
5
Mix reaction components efficiently

Use nuclease-free water for dilutions and gently pipette up and down to mix reaction components without vortexing. Keep all master mixes on ice to maintain optimal enzyme activity.

▶ 01:05
6
Add reagents and manage enzymes

Keep everything on ice and use immediately after mixing reagents. Return enzymes to the freezer promptly after use to ensure product stability.

▶ 01:23
7
Store final library samples correctly

Store finished NGS library samples in TE buffer using low-bind DNA tubes to ensure long-term stability and prevent sample degradation.

▶ 01:37

🚨 Failure Case Library (7) + Submit your own case

critical
Complete Library Preparation Failure
No library visible on Bioanalyzer or similar instrument after amplification, or library fragments remain the same size as input DNA instead of showing expected ~120 bp increase in size.
💡 4 · ✓ 4
severe
Low Library Yield with Intact Input DNA
Library yield is significantly lower than expected based on input amount, despite successful completion of all enzymatic steps.
💡 4 · ✓ 4
severe
Sample Loss During SPRI Bead Cleanup
Significantly lower library yield after SPRI bead cleanup steps, with visible reduction in final library concentration disproportionate to expected recovery rate.
💡 5 · ✓ 5
severe
Library Overamplification with Heteroduplex Formation
High molecular weight fragments appear on Bioanalyzer after PCR due to single-stranded library fragments and heteroduplex formation when PCR primers are depleted. Data quality may be compromised upon sequencing.
💡 4 · ✓ 4
moderate
Residual Adaptor or Primers After PCR
Adaptor or primer peaks visible on Bioanalyzer or similar instrument after PCR amplification, indicating inefficient cleanup or excess reagent usage.
💡 3 · ✓ 3
moderate
Excessive Adaptor Dimer Formation
Sharp 127 bp peak visible on Bioanalyzer representing adaptor dimers, reducing the proportion of desired library fragments and overall usable yield.
💡 3 · ✓ 4
moderate
Library Not the Correct Insert Size
Library fragment size distribution is shifted from expected range, with either smaller or larger inserts than intended, or discrepancy between fragment analyzer measurement and sequencer-determined insert size.
💡 5 · ✓ 5
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