Emergence of single-molecule sequencing and potential for molecular diagnostic applications

Abstract

The effective demonstration of single-molecule sequencing at scale over the last several years offers the exciting opportunity for a new era in the field of molecular diagnostics. As we aim to personalize and deliver cost-effective healthcare, we must consider the need to fully integrate genomics into decision-making. We must be able to accurately and cost effectively obtain a complete genome sequence for disease diagnosis, interrogate a molecular signature from blood for therapeutic monitoring, obtain a tumor mutation profile for optimizing therapeutic choice -each molecular diagnostic measurement utilized to better inform patient care. Would a physician or molecular pathology laboratory want to utilize a PCR process in which millions of DNA copies of a patient's nucleic acid are created when an alternative approach allowing direct measurement of the nucleic acids is possible? I would suggest not! In this article we will focus on the emergence of singlemolecule sequencing, the single-molecule sequencing methodologies in the marketplace or under development today, as well as the importance of these methods for molecular characterization and diagnosis of disease with the ultimate application for molecular diagnostics.

Next-generation sequencing in the clinic: Promises and challenges

“…The single-molecule sequencing strategy promises significant advantages over current NGS technologies in that it minimizes sample handling, reduces sample input requirements, avoids amplification-induced bias and errors, increases read length flexibility and enables accurate quantitation of nucleic acid molecules. The simplicity, sensitivity and quantitative capabilities of single-molecule sequencing make it highly promising for molecular diagnostics [11].…”

Application of Next-generation Sequencing in Clinical Molecular Diagnostics

“…The advantage of SMS lies in its ability to directly sequence single nucleic acid molecules (NA) in biological samples without amplification 12 . It can minimize sample handling, decrease sample input requirements, prevent amplification-induced bias and errors, enhance read length flexibility and enable accurate quantitation of NAs 12 .…”

“…The advantage of SMS lies in its ability to directly sequence single nucleic acid molecules (NA) in biological samples without amplification 12 . It can minimize sample handling, decrease sample input requirements, prevent amplification-induced bias and errors, enhance read length flexibility and enable accurate quantitation of NAs 12 . The Helicos Genetic Analysis System is the first commercially available SMS platform 13 In this system, poly-(A)-tailed single-stranded DNA is captured by poly-(T) oligonucleotide primers tethered to the surface of a flow cell.…”

“…The short-read length ranges from 30 bp to 35 bp at present time, with a raw base accuracy greater than 99%, and 20-28 Gbp of potential sequence reads per run in the near future 11,12 . Helicos system may also have some disadvantage.…”

Triagem de mutação no éxon 3 do gene IRF6 em indivíduos com fissura labiopalatina e agenesia dentária: padronização de protocolo para seqüenciamento de DNA genômico a partir de saliva

“…Nos últimos anos, o seqüenciamento direto de DNA genômico tem sido o método de eleição para investigar polimorfismos e mutações nas populações e relacioná-los às doenças e também contribuiu de maneira relevante para o conhecimento do genoma humano (KARGER; GUTMAN, 2009;MILOS et al, 2009;BENNET et al, 2009). Nos diversos centros de pesquisa estão sendo utilizados protocolos distintos para a preparação das amostras com vistas ao seqüenciamento direto, cada um aplicado a um tipo de plataforma de seqüenciamento (RILEY et al, 2007;JIA et al, 2009;TANG et al, 2009;SUAZO et al, 2008;BIRNBAUM et al, 2008;PEGELOW et al, 2008).…”