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Next Generation: Adapter Ligation

For a DNA fragment to be sequenced on an Illumina instrument, it first has to attach to the Illumina flow cell. The interior of the fluidics lane on each flow cell is printed, top and bottom, with a lawn of single-stranded oligonucleotides. All our DNA of interest needs to have is a complementary region on each end for clustering and sequencing to take place.

The process of adding these complementary regions - adapters - to the DNA molecules can be done through PCR for amplicon-based libraries, but is more simply done with adapter ligation, especially for whole genome or transcriptome libraries which must be randomly sheared to reach usable fragment lengths.

Fragmented DNA, particularly mechanically sheared DNA, contains variable-length overhangs, which is unsuitable for direct ligation.


sheared DNA

End repair enzymes (the exact enzymes used depends on the kit and protocol) fill in/eat away at the overhangs with their polymerase and exonuclease abilities, respectively, until the fragments are blunt-ended.


blunted DNA

An "A" is then added to the 3' ends of each fragment to improve the efficiency of the ligation reaction (ligation can be done with blunted fragments as well, but the chance of ligating two fragments to each other instead of to the adapter molecules is much higher).


A-tailing

Finally, the adapter molecules themselves are ligated to the DNA fragments. These molecules are shaped rather like the letter "Y", with a double-stranded portion able to ligate to the double-stranded DNA fragment (this portion has a "T" overhang to complement the "A" overhang on the DNA of interest), and two non-complementary single-stranded portions able to bind to the flow cell oligos. One of these single-stranded portions contains the primer for the forward sequencing read, and the other contains the primer for the reverse sequencing read. One or both may contain unique index sequences to enable pooling of multiple samples into the same flow cell.


Dual Indexing

The image above shows the creation of a dual-indexed library via adapter ligation. The Read 1 strand of each adapter contains priming sites for the forward read and the second index read, while the Read 2 strand contains priming sites for the reverse read and the first index read.

Because the DNA is denatured prior to loading and clustering on the flow cell takes place with single-stranded DNA, the non-complementarity and relative instability of the adapter-ligated fragments is not a concern. For certain protocols, the libraries are ready to be clustered at this point; for most protocols, however, a certain amount of amplification is necessary to make up for any ligation inefficiency or low levels of starting DNA.


library amplification

After PCR the "Y" shape of the adapter regions is lost and the libraries are completely double-stranded; this makes them more stable for long-term storage but doesn't affect clustering and sequencing.

At this point, the libraries are finally ready for quantification and sequencing.