Library QC & pooling
Microfluidic chip or capillary electrophoresis with an intercalating dye and sizing markers. It gives each library's size distribution and average size, and reveals dimers, bubbles and degraded or over-fragmented samples.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Bioanalyzer
Microfluidic chip or capillary electrophoresis with an intercalating dye and sizing markers. It gives each library's size distribution and average size, and reveals dimers, bubbles and degraded or over-fragmented samples.
How it differs from the standard method
- High-sensitivity assays work in roughly the 0.1–5 ng/µL range; dilute concentrated libraries first.
- Set the region to the library (e.g. 150–1000 bp) and record its average size, but deal with any dimer peak physically.
- Its concentration is less precise than fluorometry; use the trace for size and a fluorometer for mass.
1Why molarity, not mass
A sequencer counts molecules: each one that lands on the flow cell can become one cluster and give one read. Libraries are measured in mass (ng/µL), and the same mass of a long library contains fewer molecules than of a short one.
Sizing trace
Capillary or chip electrophoresis gives the size distribution, the average size of a chosen region, and shows dimers and over-amplification.
Fluorometry
A dsDNA dye measures mass accurately at low concentrations; absorbance over-reads dilute samples and counts contaminants.
qPCR
Adapter-specific primers count only molecules with both adapters, the ones that can actually form clusters.
2Reading a library trace
A good library is one smooth peak in the expected range, flanked by the lower and upper markers. A sharp peak at 120–140 bp is adapter or primer dimer: it must be cleaned away before pooling, because dimers cluster efficiently whether or not you include them in the sizing region.
3qPCR quantification
Library qPCR amplifies a dilution of the library alongside a series of DNA standards of known concentration that carry the same adapters. Cq falls by about 3.3 cycles for every 10-fold more template, so the standards give a line and the sample's Cq is read off it.
4Pooling
- Convert each library to nM with its own trace size and a fluorometric concentration (or use qPCR values).
- Dilute every library to the same molarity (e.g. 2–4 nM) in buffer with a little detergent, then combine equal volumes.
- Avoid pipetting below ~1 µL; dilute first instead.
- Re-clean any library with a dimer peak before it goes in.
- Quantify the final pool by qPCR before denaturing and loading.
Key terms
Practise these as flashcards →The mass-weighted mean length of the part of the trace you select; it is used for the mass-to-molar conversion.
Molecules per volume (nM); what loading concentration and pooling are set in.
A pool in which each library contributes the same number of molecules, so each gets a similar number of reads.
Multiplying a qPCR result by standard size ÷ library size to correct for dye signal per molecule.
Cq against log concentration for the standards; slope about −3.3 at 100% efficiency.
A qPCR well with no library, showing that primers and reagents are free of contaminating library.
Coefficient of variation of the fraction of reads per sample; a common measure of pool balance.
When to use it
- Every library, before pooling: a sizing trace plus a concentration.
- qPCR quantification before loading a pool, and whenever accurate cluster density matters (patterned flow cells, expensive runs).
- Whenever library sizes in a pool differ, so each needs its own size in the molarity calculation.
Compared with alternatives
The step before: problems seen on the trace (dimers, short inserts, bubbles) are fixed there.
Where the pool goes; loading concentration follows from the qPCR molarity.
General DNA quantification: absorbance, fluorometry and fragment analysis for any DNA sample.
The same standard-curve qPCR, applied to library molecules rather than a gene target.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Microfluidic chip or capillary electrophoresis with an intercalating dye and sizing markers. It gives each library's size distribution and average size, and reveals dimers, bubbles and degraded or over-fragmented samples.
- High-sensitivity assays work in roughly the 0.1–5 ng/µL range; dilute concentrated libraries first.
- Set the region to the library (e.g. 150–1000 bp) and record its average size, but deal with any dimer peak physically.
- Its concentration is less precise than fluorometry; use the trace for size and a fluorometer for mass.