SMRT™ Biology SAMPLE PREP

SMRTbell™ Template Preparation

Our SMRTbell sample preparation method is an innovation that creates a circularized template for use with multiple sequencing protocols. Using one streamlined protocol you can create libraries of varying insert length depending on the needs of your application. For example, due to our long read lengths and strand displacing enzyme, our circular consensus sequencing method reads templates multiple times to achieve unprecedented accuracy on a single molecule. This type of circular consensus accuracy is novel, as it is fundamentally impossible to achieve with an ensemble-based approach. This method also offers advantages for the discovery and confirmation of rare variants. Furthermore, this approach provides reads on both the forward and reverse strands, providing more insights into the source and nature of genetic changes.

Unlike other circularization methods that are complicated, waste a significant amount of sample, and are limited in the insert size, SMRTbell sample prep is straightforward and efficient. Insert size is not a constraint because universal hairpin adapters are ligated onto double stranded DNA. Overall, you can complete the sample prep in 3-6 hours with minimal hands on time, and go from sample prep to sequence result in under one day.

SMRT sequencing does not require routine amplification like other sequencing methods, and it is simple to convert a variety of sample types (e.g., purified genomic DNA, BACs, cDNA libraries or PCR products) into a SMRTbell sample using familiar techniques.

Figure 18
Figure 18. SMRT™ sequencing sample preparation workflow.
The input sample is first fragmented to the desired size. The ends are repaired and the hairpin structures are ligated to the ends of each fragment. A size selection and purification step selects those fragments with the adaptors attached to both ends. The SMRTbell templates then can go through the sequencing reaction. A strand displacing polymerase enzyme opens the SMRTbell into a circular template and can generate independent reads, both forward and reverse of the same DNA molecule. The quality score increases linearly with the number of times the molecule is sequenced.

 
Next: SMRT DNA Sequencing Protocols for Standard, Circular Consensus, and Strobe Sequencing.