Overview:

Mate-pair libraries are used for deep sequencing of a whole genome.  They may also be useful for some other applications, such as sequencing a selected region of the genome, where detection of structural genomic changes is desired.   The lab process begins with total genomic DNA, which is fragmented to a chosen size range – we have used 1,500 base pairs as a target size.  A mate-pair library is constructed.  Each molecule within the library contains ~100 base pairs from one end of the genomic fragment as well as ~100 base pairs from the other end of that genomic fragment.  Each of the two 100 base pair fragments is downstream from a different primer sequence (the steps in this procedure are described in more detail in the “Library construction” section directly below).  One primer is used to direct the sequencing of 50 bases of one of the fragments; subsequently, the second primer directs the sequencing of 50 bases of the other fragment (an overview of the sequencing chemistry can be found by following this Link). Point mutations and small insertions and deletions can be detected in the resulting sequences.  Moreover, “mate” sequences that are not located within an expected distance from each other alert us to the presence of significant structural aberrations, including translocations, inversions, deletions and insertions/amplifications, in specific regions of the genome.  In our pilots, ~45% of the ~300 million reads/slide (2 x 50 bases per bead) map uniquely to the genome.  Since two slides can be processed per run of the instrument, approximately 27 billion uniquely mapping bases per run are generated. It is expected that this number will roughly double by the end of 2009.

Overview:

Fragment libraries are simpler to construct than mate-pair libraries, and do not require high molecular weight DNA.  The libraries constructed from them are sequenced (50 bases at this time) from one end only (at this time).  Total genomic DNA, or DNA that has been enriched for selected regions of the genome, can be used as starting material.