Sequencing Structurally Complex DNA Regions with Sanger and Pacific Biosciences Technologies
Sanger sequencing remains a valuable tool for determining nucleotide sequences of individual DNA molecules. For large genome projects, Sanger sequencing has been supplanted by NextGen DNA sequencing technologies. However, Sanger sequencing is still widely used for small-scale projects and for obtaining contiguous reads of 800-1000bp.
Although Sanger sequencing is very robust and routinely gives 800-1000bp, in many cases it fails to give high quality reads through complex DNA regions, e.g. DNA with high GC rich content, complex sequence repeats, hairpin structures, and long homopolymer stretches.
Pacific Biosciences (PacBio) third generation technology provides an approach that is different from Sanger and second generation sequencing technologies, and has the potential to overcome common DNA sequencing challenges by providing significantly longer reads (average readlength >4,000bp). A recent study within the Genomics division of ICBR reports on nucleotide sequence data for a plasmid template, containing inverted tandem repeats with long Poly G and Poly C and regions with strong hair pin structures, using a specially modified Sanger sequencing approach and then also PacBio sequencing.
Different methods were tested to sequence ITR and CBA promotor region by Sanger sequencing. Only the use of sequence enhancer buffer A showed some improvement in data, but overall the sequence quality was very poor. PacBio sequencing data from ~4kb fragment with ITR and CBA promotor demonstrates that it is a promising platform to sequence structurally complex DNA regions. This technology is not adversely influenced by the presence of Poly G, Poly C, or highly rich GC rich regions.