NGS Library Complexity Calculator
Calculate library complexity and PCR duplicate rate for NGS sequencing. Estimate distinct molecules using Lander-Waterman theorem. Assess whether more sequencing depth will yield new information.
🧬 Genomics📐 distinct = total x (1 - e^(-coverage))🧬 Biology
Total reads sequenced (millions)
Duplicate rate (%)
Input material (ng)
Please enter valid values.
Formula & Reference
| Variable | Symbol | Formula | Units |
|---|---|---|---|
| NGS Library Complexity Calculator | — | distinct = total x (1 - e^(-coverage)) | molecules |
Step-by-Step Examples
Example 1
Low Complexity Library
50M reads, 60% duplicate rate.
- Unique reads = 50M x 0.40 = 20M unique
- Estimated complexity = 20M / 0.40 = 50M molecules
- Library saturated — more sequencing won't help
- Need more input material or new library
✓ Low complexity, don't sequence deeper
Example 2
Good Complexity Library
100M reads, 15% duplicates.
- Unique reads = 100M x 0.85 = 85M unique
- Complexity estimate = 85M / 0.85 = 100M molecules
- Duplicate rate good — library not saturated
- More sequencing will yield new information
✓ Good complexity, can sequence deeper
Example 3
Targeted Panel
10M reads, 40% duplicates, 50Mb target.
- Unique reads = 6M
- For clinical panel, 30-50% dups acceptable (high coverage of targets)
- Check on-target rate and coverage uniformity
✓ 40% duplicates acceptable for targeted panel
Real-World Applications
WGS Quality Control
Library complexity determines how many unique molecules represent the genome.
RNA-Seq Saturation
Library saturation analysis shows whether more reads will discover new transcripts.
ChIP-Seq
Low complexity in ChIP-seq libraries indicates poor immunoprecipitation efficiency.
Clinical NGS
High duplicate rates in liquid biopsy require special consideration for ctDNA detection.
Common Mistakes to Avoid
⚠️
Not checking duplicate rate before deep sequencing
If library is saturated (>60% duplicates), additional sequencing is wasted. Check duplicate rate on a shallow pilot run first.
⚠️
Confusing sequencing depth with library complexity
You can sequence the same library to 100x depth, but if there are only 10 distinct molecules, you're just re-reading the same 10 molecules.
⚠️
Using all PCR cycles regardless of input
More PCR amplification = more duplicates. With high-input DNA: use 5-8 cycles. With low-input: 12-18 cycles. Use PCR-free protocol when possible for WGS.
Frequently Asked Questions
What is PCR duplication? ▾
During library preparation, PCR amplification creates copies of each original DNA molecule. These copies are sequencing artifacts — they look the same but carry no extra information.
What is acceptable duplicate rate? ▾
WGS: <30%. RNA-seq: <40-50%. ChIP-seq: <40%. Targeted sequencing: up to 60% can be acceptable if coverage is sufficient. Liquid biopsy: use UMI to handle high duplicates.
What is UMI? ▾
Unique Molecular Identifier — random barcode added to each DNA molecule before PCR. Allows distinguishing true duplicates (same UMI) from independent fragments (different UMI). Standard in liquid biopsy.
How do I reduce duplicates? ▾
Use more input DNA, reduce PCR cycles, use PCR-free protocol for WGS, optimize library prep. For targeted sequencing, some duplicates are expected due to limited complexity of capture.
What is library saturation? ▾
When most unique molecules have already been sequenced and additional sequencing mainly picks up PCR duplicates. Assessed by saturation analysis: plot new unique reads vs total reads — flattening = saturated.