The preliminary objective is to fragment the DNA to give suitably sized DNA fragments, and then attach to them common sequences that can be recognized by universal primers for amplification and DNA sequencing. Fragmentation of the DNA is usually achieved using a mechanical method. Sonication uses sound energy to agitate the DNA molecules in solution; hydrodynamic shearing forces break the DNA at random locations. Alternatively, nebulization randomly shears DNA by using pressurized gas to force the DNA through a small hole in a nebulizer unit. Conditions are chosen to prepare small DNA fragments within a desired size range.
The resulting DNA fragments have different lengths of overhanging 5′ and/or 3′ ends but can be converted to blunt-ended DNA using a DNA polymerase and 3′ exonuclease (Figure 1A). Blunt-ended DNA fragments are phosphorylated at the 5′ end, and then are usually A-tailed at the 3′ end to facilitate ligation to a double-stranded adaptor oligo nucleotide designed to have an overhanging T.
Fig1. Preparation and amplification of DNA libraries for high-throughput DNA sequencing. (A) DNA fragment preparation. The DNA is randomly fragmented. The resulting fragments are made blunt-ended by digesting 3′ overhangs with a 3′ exonuclease and by “filling in” at overhanging 5′ ends (extending the complementary strand using DNA polymerase). Blunt-ended fragments are 5′ phosphorylated using polynucleotide kinase and are often then “A-tailed” using Taq polymerase to add a nontemplated adenine to the 3′ end. (B) Amplification using a forked adaptor oligonucleotide. A forked adaptor oligonucleotide is designed to be partially double stranded (open boxes) but has distinctive sequences (labeled 1 and 2 here) that will provide target sequences for forward and backward primers in the sequencing reaction. To facilitate ligation, one of the two oligonucleotides is often designed to have an additional T (resulting in a 3′ T overhang). Thereafter, primers for adaptor sequences 1 and 2 are used to amplify the DNA fragments. The primers, however, are asymmetric. One primer (primer 1 here) has a sequence complementary to one of the unique adaptor sequences, and base-pairs immediately with its target. However, primer 2 has the same sequence (not the complementary sequence), as part of the other adaptor sequence (and so cannot bind initially). After one round of DNA synthesis (by primer 1 only), a desired product is formed with the two different, flanking double-stranded adaptor sequences, and can now be amplified after both primers 1 and 2 bind. (C) An example of an Illumina paired-end forked adaptor (the asterisk signifies a phosphorothioate bond that is resistant to 3′ exonuclease).
The adaptor oligonucleotide provides defined target sequences to allow binding of complementary primers to enable clonal amplification of individual DNA fragments, and can be used to allow sequencing of both ends of each DNA fragment. Forked adap tors are typically used. These Y-shaped adaptors are designed to have complementary sequences at one end (which form a double-stranded stem) and two unrelated and non complementary sequences at the other end (which form single-stranded arms). The amplification process allows individual DNA fragments to be flanked by two different sequences to which sequencing primers can subsequently bind for forward and reverse strands (Figure 1B and C).
