Library Preparation

The SOLiD system supports sample preparation for fragment or mate-paired libraries with insert sizes ranging from 60-110bp and 600-6kbp, respectively. Depending on the information needed, libraries with a range of insert sizes are created to enable more precise characterization of structural variation across the genome or targetted sequence. Either for a fragment or Mate-paired library, a template (RNA, DNA, cDNA, or PCR prodcut) is sheared by sonication (Covaris S2) or physical disruption (HydroShear).

mate pair

 

The sheared template is end-repaired, purified and ligated to adaptors to obtain a fragment library. These adaptors, P1 and P2, are used to amplify copy number of the genomic target(s). After the library is amplified, a size-selection is performed in Agarose or PAGE gels in order to obtain the desired fragment size containing the template ligate to adaptors.

frag prep mare prep 1

A few additional steps are performed after shearing and end-repairing the template to obtain a mate-paired library. A mate-paired library consists of a pair of DNA fragments that are “mates” because they originated from the two ends of the same genomic DNA fragment. The sheared end-repaired template is methylated and caped with EcoP15I CAP adapters. EcoP15I CAP adapters connect the DNA mate-pairs together through a biotinylated internal adapter resulting in DNA circularization. EcoP15I cleaves 25-27 bp away from the unmethylated enzyme recognition sites in the CAP linker, yielding mate-paired genomic DNA attached to both sides of the internal adapter. P1 and P2 are then ligated to the ends of the mate-paired library for subsequent amplification by PCR and enrichment of amplified template.

mate prep 2