Library construction
Break DNA into random pieces of a chosen size, either physically (focused acoustic shearing, bath sonication) or with a fragmentase enzyme mix whose incubation time sets the size.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Sonication / enzymatic fragmentation
Break DNA into random pieces of a chosen size, either physically (focused acoustic shearing, bath sonication) or with a fragmentase enzyme mix whose incubation time sets the size.
How it differs from the standard method
- Acoustic shearing is reproducible and nearly sequence-independent; time and power set the size, with diminishing returns.
- Enzymatic fragmentation needs no instrument and is easy to automate, but is sensitive to input amount, EDTA and temperature, and has mild sequence preferences.
- Either way, check a test aliquot on a size trace before committing the whole sample.
1What a library is
A short-read sequencer can only read molecules of a few hundred base pairs that carry known sequences at both ends. Library construction turns DNA (or cDNA) into exactly that: fragments of a chosen size, each flanked by adapters that hold the flow-cell binding sites, the sequencing primer sites and a sample index.
Insert size
Set by fragmentation. It should be longer than the read length, or reads overlap and run into the adapter.
Complexity
The number of distinct adapted molecules. PCR copies them but cannot add new ones, so complexity caps how deeply a library is worth sequencing.
Purity
Adapter and primer dimers carry no insert but cluster very efficiently, so they take reads.
2Three routes to adapted fragments
- Shear, repair, ligate
- Sonication or an enzyme mix breaks DNA into random fragments. The ragged ends are filled in and phosphorylated, a single A is added to each 3′ end, and Y-shaped adapters with a T overhang are ligated on. Flexible and low-bias; it takes several steps.
- Tagmentation
- A transposase pre-loaded with adapter sequences cuts DNA and pastes the adapters in a single step. The DNA-to-enzyme ratio sets the fragment size, so the input must be measured accurately. The enzyme leaves short gaps that a first extension step fills.
- Index PCR
- A few PCR cycles with primers that complete the adapters and add the sample indexes. It makes enough material to quantify and load, but every extra cycle adds bias and, past the plateau, artefacts.
3How libraries fail
Most failures show on the final size trace. A sharp peak at 120–140 bp is adapter or primer dimer, from too much adapter for the input or a clean-up that kept short DNA.
Key terms
Practise these as flashcards →The sample-derived part of a library molecule, between the two adapters.
A partly double-stranded adapter whose unpaired arms make the two ends of each library molecule different, so every insert can be read from both sides.
Adding a single 3′ adenine to blunt fragments so they ligate to T-overhang adapters and not to each other.
An enzyme that fragments DNA and inserts adapter sequences at the same time.
Carboxyl-coated magnetic beads that bind DNA in PEG and salt; the bead-to-sample ratio sets the size cut-off.
The number of distinct molecules in a library; it sets the duplicate rate at a given sequencing depth.
Index pairs in which neither index is shared with another sample, so swapped (hopped) index reads can be discarded.
Heteroduplexes formed after over-amplification that run anomalously large on a size trace.
When to use it
- Before any short-read sequencing of genomic DNA, cDNA, ChIP or ATAC material, or amplicons that do not already carry adapters.
- Tagmentation when input is low and speed matters, and the DNA amount can be measured well.
- Mechanical shearing and ligation when insert size must be tightly controlled or sequence bias kept low, or for PCR-free libraries from high inputs.
Compared with alternatives
The next step: sizing, quantifying and pooling finished libraries at equal molarity.
Where the library is used; loading and cluster density depend on how well the library was built and quantified.
Long-read libraries keep fragments long (kilobases to tens of kilobases) and are usually not amplified.
Index PCR is ordinary PCR with long tailed primers; amplicon libraries can be built entirely by two rounds of PCR.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Break DNA into random pieces of a chosen size, either physically (focused acoustic shearing, bath sonication) or with a fragmentase enzyme mix whose incubation time sets the size.
- Acoustic shearing is reproducible and nearly sequence-independent; time and power set the size, with diminishing returns.
- Enzymatic fragmentation needs no instrument and is easy to automate, but is sensitive to input amount, EDTA and temperature, and has mild sequence preferences.
- Either way, check a test aliquot on a size trace before committing the whole sample.