Impact of DNA Quality on PacBio RSII Read Lengths

Obtaining the longest Next Generation Sequencing (NGS) reads possible facilitates de novo genome assembly.  Currently, the Pacific Biosciences RSII system leads the industry with respect to maximum possible NGS read lengths.  Libraries must be prepared from high quality gDNA In order to achieve the best results with the RSII system, high quality gDNA must be used when preparing templates for sequencing.

Amplicon Express specializes in preparation of High Molecular Weight, NGS-grade gDNA for a variety of applications.  This study was performed to evaluate the performance of genomic DNA prepared by Amplicon Express on RSII read length.

Genomic DNA was isolated from E. coli K12 strain MG1655S by Amplicon Express and another commercial supplier.  Large insert NGS libraries from both gDNA sources were prepared and sequenced in parallel at Pacific Biosciences. 

Pulse-field gel QC indicated that the SMRTBell libraries prepared from gDNA isolated by Amplicon Express had an overall larger insert size.  RSII sequencing data demonstrated a 15% increase in read length for the Amplicon Express library.  A similar 15% improvement in polymerase survival was seen for this library. Yield and loading of the libraries were comparable.  Thus, libraries prepared from gDNA prepared by Amplicon Express had larger insert sizes, and less internal polymerase-terminating damage.

Please visit our PAG poster for details on the DNA protocol/services optimized to get the best data possible from Pacific Biosciences’ RSII system for your project.