Getting the Most Out of Your PacBio® Libraries with Size Selection

PacBio® RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing.   The recently launched P5-C3 chemistry generates even longer reads with an N50 >10,000 bp, best for scaffolding and spanning structural rearrangements.  With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell^™library itself. 

Size selection of a high-quality sheared 20 kb library using the BluePippin™ System has been demonstrated to increase the N50 read length by as much as 5 kb with C3 chemistry.  Even on degraded samples, it is possible to use size-selection strategies such as BluePippin™ selection or a stringent AMPure® selection cutoff to recover long reads suitable for genome sequencing.  The selection of chemistries, P4-C2 verses P5-C3, is highly dependent on the final size distribution of the SMRTbell library and experimental goals.   

PacBio’s long read lengths also allow for the sequencing of full-length cDNA libraries at single-molecule resolution.  However, longer transcripts are difficult to detect due to lower abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Without size selection, most sequenced transcripts are between 1-1.5kb. Size selection dramatically improves the uniformity of sequenced transcripts lengths, particularly for those > 3kb.

This poster will demonstrate the benefits of size-selection strategies and chemistry selection to help improve experimental design that maximizes SMRT® Sequencing performance for gDNA and cDNA applications.