Leading to a Higher Level of de novo Genome Assembly: PacBio RS Sequencing

The PacBio RS (Pacific Biosciences Inc.) is a 3rd Generation Sequencer accomplishing single molecule sequencing (SMRT technology). This sequencing technology has significant difference to those of the second generation: library preparation does not include any PCR step, which usually amplifies GC-rich sequences with low efficiency. This should lead to more evenly covered genomes and no dependence to base composition. Read length is much longer, leading to significantly longer contigs after the assembly of the sequencing reads. A disadvantage of the PacBio RS platform is the error rate of about 15%. In contrast to other technologies PacBio RS has a much lower sequence bias. The errors are distributed randomly, giving the possibility to use higher coverage for more accuracy. “Circular consensus sequencing reads" (CCS), in which the circular fragments have been sequenced several rounds, can be used to achive higher accuracy. Alternatively, the error rate can be lowered by combining PacBio RS data with data from other sequencing technologies.  

A comparison of assemblies of different genomes produced with data from HiSeq, GS FLX and PacBio RS reads shows significant differences in assembly quality. This new and innovative sequencing approach using PacBio RS is suitable to simplify the analysis of unknown genomes and will improve the analysis of genomic complexity. Also, using long reads, transcript coverage can be improved or covered completely, which makes the analysis of splice variants easier, help to discover genetic diseases and also improve genome assemblies.

In addition, examples for direct analysis of modified nucleotides will be shown.