Sequencing Paired Reads using True Single Molecule Sequencing (tSMS)™ Technology

Abstract

Single molecule DNA sequencing provides novel methods for interrogating DNA molecules. For example, genomic rearrangements such as insertions, deletions, and inversions that are often associated with cancers or variations within the transcriptome of specific genes can be difficult to detect with conventional sequencing strategies. Paired reads sequencing, where a spacer is inserted between two single molecule sequencing reads, offers a more viable method for detecting genomic rearrangements. We have developed a paired reads strategy using True Single Molecule Sequencing (tSMS)™ in which a large number of individual templates of DNA were analyzed using a proprietary form of sequencing-by-synthesis. To create paired reads DNA strands are attached to a surface and sequenced-by-synthesis for a known number of cycles. A spacer was then added to the DNA strands in a controlled manner and then sequencing by synthesis continued for the same number of cycles. Data on test oligonucleotides of known length and sequence demonstrate the viability of the technique and our ability to control the length of the spacer between the two reads on an individual strand. We have now extended our Paired Reads technique to biological samples, initially with a 12kb PCR product encompassing the CETP gene to demonstrate our ability to sequence the whole gene product and identify mutations which have been inserted into the CETP reference. Finally we have utilized this novel method to examine a human placental transcriptome cDNA library to demonstrate the ability to span exon boundaries.