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J Biomol Tech. 2010 September; 21(3 Suppl): S28.
PMCID: PMC2918047

Breaking Barriers through Single Molecule, Real Time Sequencing

Abstract

RP-26

This presentation is an overview of the novel applications enabled by SMRT™ sequencing. In addition to describing the performance specifications of the instrument which will ship later this year, we will give an overview of useful characteristics of the system: long readlength, fast time-to-result, high accuracy through circular consensus, robust immunity from systematic error and sequence content bias, and richer data. These features will then be highlighted through application to model systems. Transcript sequencing has previously been limited to messages less than ~1100 bases in length for sequencing in one contiguous read. We will present a >3,000-base transcript sequenced in a single read, encompassing an entire aberrant cancer transcript. We will apply strobe sequencing — a method to further extend the physical coverage of individual reads — to structural variations in complex genomes demonstrating individual reads to10,000 bases. The fast time-to-data will be exploited in a study of H1N1 influenza virus in which we traverse the entire process from sample arrival to variant identification in less than 10 hours. We will show a completed sequence of a GC-rich organism relevant to biofuel generation enabled by our long read technology. We will show how the rich set of kinetic information allows direct detection of various forms of modified bases such as 5-methylcytosine, 5-hydroxymethyl cytosine, N6 methyladenosine as well as a host of other medically and biologically important modifications. We will culminate by demonstrating applications of the SMRT detection system to follow SMRT DNA sequencing. First, we will show sequencing of RNA directly in real time without conversion to cDNA through the use of an RNA-templating polymerase, and second we will show the first real-time detection of translation with amino acid resolution by a ribosome immobilized in a ZMW.


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