You finally finish your precious library prep, send it off for sequencing... and then comes the nightmare: the QC report says “FAILED.” What happened? Wide peaks, messy peaks, short fragments, smears, adapter dimers, contamination, or just not enough concentration?
So, what does a high-quality sequencing library look like? And more importantly—how do you improve your library success rate?
Library QC 101: What Really Matters?
There are two key checkpoints in library QC:
1. Library Concentration
This is typically measured using Qubit dsDNA assays, which rely on fluorescent dyes that specifically bind to double-stranded DNA. These dyes emit fluorescence only when bound to target molecules, providing highly accurate quantification.
✅ Most sequencing platforms require: Library concentration ≥ 2 ng/μL
But beware—just passing the concentration test doesn’t mean your library is sequencing-ready. If your size distribution is off, the actual usable library concentration may be much lower than reported.
2. Library Size Distribution
The most commonly used tool here is the Agilent Bioanalyzer, which leverages microfluidic capillary electrophoresis.
Here’s how it works:
- A voltage drives your sample through tiny etched microchannels.
- DNA fragments are separated by size as they travel.
- Fluorescent dye binds to double-stranded DNA and is excited by a laser to produce detectable signals.
- The result: an electropherogram showing the distribution of DNA fragment sizes—your library’s fingerprint.
You’ll also often estimate molarity using:
Library molarity (pmol) ≈ Mass (ng) / [0.66 × Average fragment size (bp)]
✅ What Does a “Good” Library Look Like?
For PE150 sequencing (paired-end 150 bp), a high-quality library should meet the following criteria:
- Main peak between 300–600 bp
- Smooth, bell-shaped curve resembling a normal distribution
- No visible adapter dimers or primer-dimer peaks
- Minimal background noise or secondary peaks
Figure 1. Representative Electropherogram of a Qualified Sequencing Library
Adapter Dimers, Smears, and Mystery Peaks? Here's What Your Electropherogram Is Telling You
You've just QC'd your library, and the electropherogram looks... suspicious. There's a tiny peak where it shouldn’t be, a big fat bump that spans too wide, or even multiple peaks screaming contamination. Let’s walk through the most common library QC failures, why they happen, and—most importantly—how to fix them.
1. Adapter Contamination / Short Fragment Pollution (15–270 bp)
This is by far the most common reason libraries fail QC. Residual adapters or short fragments tend to cluster preferentially during sequencing, hijacking valuable space in the flow cell. The result? Lower overall yield and reduced usable data.
If the short fragment area exceeds 3% of the total library peak, the library may be rejected.
Possible causes:
Degraded samples – Fragmented or smeared inputs lead to more short junk.
→ Use fresh samples and optimize fragmentation conditions.
Improper bead selection ratio – Size selection is key.
→ Adjust cleanup ratios to better exclude unwanted fragments.
Excess adapters added – Overloading adapters leads to inefficient purification.
→ Dilute adapters based on input amount to avoid leftover dimers.
Figure 2. Library Contamination with Adapter Dimers and Short Fragments
2. Trailing Peaks: When Your Library Doesn’t Taper Off Right
One of the most common signs of a problematic library on an electropherogram is “tailing”—that is, the main peak doesn't return cleanly to baseline and looks asymmetrical or smeared on one side.
If the trailing region accounts for more than 40% of the total distribution, the library may be flagged as having a tailing issue and considered QC-failed.
Possible Causes of Tailing:
1)Improper gel excision range
If you’re using gel-based size selection, choosing a too-wide or off-target fragment range can lead to unwanted size carryover.
2)High salt concentration in the reaction mix
Residual salts from extraction or cleanup can affect polymerase performance and cause fragment quality issues.
→ Consider an additional nucleic acid purification step before library preparation.
3)Over-amplification during PCR
Too many cycles or insufficient primers can cause non-specific amplification, leading to low yield and smeared peaks.
→ Optimize primer concentrations and avoid excessive PCR cycling.
Figure 3 Electropherogram Showing a Tailing Library Profile
3. Wide Peaks = Fragmentation Issues
When your library looks "chubby," with a peak that starts and ends beyond the expected size range, it's often a fragmentation problem. Ideally, the peak should be tight and centered between ~300–600 bp (for PE150).
Possible causes:
Suboptimal fragmentation conditions – Too hot, too long, or too much enzyme.
→ Tune your fragmentation settings to your target size.
Incorrect bead cleanup ratio –
→ Calibrate your size selection protocol to tighten the peak.
Low-quality DNA/RNA input – Degradation or smeared bands on gel.
→ Use intact, high-quality starting material.
Figure 4. Abnormal Fragment Distribution: Broad Peak
4. Multiple Peaks / Mystery Peaks (Double Peaks or Non-target Peaks)
If your electropherogram shows more than one peak—especially non-target fragment sizes—you may be dealing with contamination or poor size selection.
Possible causes:
Sample cross-contamination – From shared tips, tubes, or pipetting errors.
→ Check your lab practices and change tips between samples.
Inadequate size selection –
→ If cleanup conditions are off, small and large fragments may sneak in.
Figure 5 Abnormal Library Profile: Mixed Peaks
How to Avoid All This? Start With Better Kits.
Many of these issues stem from:
- Low-quality or degraded DNA/RNA
- Inconsistent fragmentation
- Inefficient adapter ligation
- Inadequate purification
That’s why choosing a robust and well-optimized library prep kit is so important.
✅ High-quality DNA and RNA library preparation kits, like Yeasen’s 12927 and 12972 series, are designed to minimize common failure points and deliver smooth, symmetric, high-yield libraries—with fewer surprises on the Bioanalyzer.
May Your Peaks Be Clean and Your Clusters Be Plenty
QC pain is real—but it’s fixable. With the right protocols, proper controls, and high-performance reagents, you can get your libraries sequencing-ready with confidence.
Related Products
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Category |
Name |
Cat. No. |
Note |
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DNA Lib Prep Kit |
- |
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12972ES |
- |
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RNA Lib Prep Kit |
12308ES |
Strand specific & Non Strand specific |
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|
12340ES |
Strand specific |
|
|
|
12341ES |
Non Strand specific |
|
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|
12257ES |
Human/Mouse/Rat |
|
|
|
12254ES |
Plant |
|
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12629ES |
- |