Principles for construction of mate-pair sequencing libraries. (a) Preparation of Illumina mate-pair libraries. Fragments are end-repaired using biotinylated nucleotides (1). After circularization, the two fragment ends (green and red) become located adjacent to each other (2). The circularized DNA is fragmented, and biotinylated fragments are purified by affinity capture. Sequencing adapters (A1 and A2) are ligated to the ends of the captured fragments (3), and the fragments are hybridized to a flow cell, in which they are bridge amplified. The first sequence read is obtained with adapter A2 bound to the flow cell (4). The complementary strand is synthesized and linearized with adapter A1 bound to the flow cell, and the second sequence read is obtained (5). The two sequence reads (arrows) will be directed outwards from the original fragment (6). (b) Preparation of Roche 454 paired-end libraries (these are called paired-end, but are based on the same principles as the mate-pair libraries in the other technologies). Original fragments (1) are end-repaired with unlabeled nucleotides, and biotin-labeled circularization adapters (CA) are ligated to the fragment ends (2). After circularization (3), fragmentation and affinity purification, library adaptors (LA1 and LA2) are ligated to the new fragment ends (4) and the fragments are amplified on beads by emulsion PCR. One single sequence read that covers the two original ends and the internal adapter is generated (5). Adapter sequence is removed in silico, and the sequence is split into two reads, which both have the same orientation (6). (c) Preparation of SOLiD mate-pair libraries. Steps 1 to 4 are analogous with preparation of Roche 454 paired-end libraries, with a biotin-labeled internal adapter (IA) and two sequencing adapters (P1 and P2). Sequencing is performed with two different primers, complementary to the P1 adapter and internal adapter, respectively (5). The resulting reads will have the same orientation (6).