P0003 Novel Chimera-Free, High Efficiency Library Preparation for NGS Platforms - NxSeq Technology

Curtis Knox , Lucigen Corporation, Middleton, WI
Michael J. Lodes , Lucigen Corporation, Middleton, WI
Cheng-Cang Wu , Lucigen Corporation, Middleton, WI
Svetlana Jasinovica , Lucigen Corporation, Middleton, WI
David Mead , Lucigen Corporation, Middleton, WI
The efficiency of Next Generation Sequencing (NGS) depends on the quality of libraries used.    Existing NGS sample preparation kits are not optimized for A-tailing, resulting in libraries that are not efficiently ligated to T-tailed adapters, are contaminated with chimeras and concatemers, or require lengthy protocols to finish.  In addition, because enzymes such as Taq DNA polymerase add non-templated purines (both A and G residues) to the 3’ end of templates, these kits produce both A- and G-tailed templates, which also reduces the ligation efficiency to T-tailed adapters.  We have developed an optimized system that repairs template ends and maximizes A-tailing and subsequent ligation to T-tailed adapters.  Optimization was accomplished with an assay that allows one to quantify A- and G- tailing of target DNA by ligation to T- and C- tailed fluorescent adapters.  Our 90 bp target DNA consists of a mixture of random 3’ ends that must be end repaired and phosphorylated before self ligation is possible.  A-tailing prevents self ligation and chimera formation.  The results demonstrate an essentially chimera free target with over 80% efficiency of T-tailed adapters ligated in a single step compared to significant concatemer formation and low T-tailed adapter ligation (7% to 48%) with other systems. We confirmed the efficiency of our method by constructing and sequencing Ion Torrent random shear genomic libraries.  Sequence results show that Lucigen reagents produce a more complex, less biased library with fewer chimeras and concatemers than libraries produced with competitor’s kits.