Single Molecule Real Time Sequencing - Application

Application

Single molecule real time sequencing will be applicable for a broad range of genomics research, namely:

• De novo genome sequencing: Read lengths from the single molecule real time sequencing are comparable to or greater than that from the Sanger sequencing method based on dideoxynucleotide chain termination. The longer read length allows de novo genome sequencing and easier genome assemblies. SMRT sequencing has been demonstrated for de novo genome sequencing in publications analyzing the E. coli outbreak in Germany in 2011 and in the cholera outbreak in Haiti in 2010, both in the New England Journal of Medicine. Scientists are also using single molecule real time sequencing in hybrid assemblies for de novo genomes to combine short-read sequence data with long-read sequence data. In 2012, several peer-reviewed publications were released demonstrating the automated finishing of bacterial genomes, including one paper that updated the Celera Assembler with a pipeline for genome finishing using long SMRT sequencing reads.

• Resequencing: A same DNA molecule can be resequenced independently by creating the circular DNA template and utilizing a strand displacing enzyme that separates the newly synthesized DNA strand from the template. Scientists from Pacific Biosciences, the University of California, and other institutes used this circular consensus approach with SMRT sequencing to prove the validity of activating internal tandem duplication mutations in FLT3 as a therapeutic target in acute myeloid leukemia. Their findings were published in the journal Nature in April 2012. In August 2012, scientists from the Broad Institute published an evaluation of SMRT sequencing for SNP calling.

• Methylation detection: The dynamics of polymerase can indicate whether a base is methylated. Scientists demonstrated the use of single molecule real time sequencing for detecting methylation and other base modifications in a series of peer-reviewed papers in 2011. In 2012 a team of scientists used SMRT sequencing to generate the full methylomes of six bacteria, reporting their findings in the Nucleic Acids Research journal.