Abstract
BackgroundMassively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour.MethodsHere, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run.ResultsWe observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing.ConclusionsThese results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.
Highlights
Parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application
We previously developed and reported Hi-Plex, a streamlined highly-multiplexed PCR approach for Massively parallel sequencing (MPS) library preparation, using DNA derived from both lymphoblastoid cell line and formalin-fixed, paraffinembedded tumour tissue [1]
We recently demonstrated that Hi-Plex using hybrid adapter primers can produce libraries suitable for both the Ion Torrent (PGM and Proton instruments, Life Technologies, Carlsbad, CA, USA) and TruSeq (MiSeq and HiSeq instruments, Illumina, San Diego, CA, USA) systems, which currently represent the two most commonly used MPS chemistries [2]
Summary
Parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. There has been considerable discussion regarding how massively parallel sequencing (MPS) can optimally be applied in the context of clinical genetics services. We previously developed and reported Hi-Plex, a streamlined highly-multiplexed PCR approach for MPS library preparation, using DNA derived from both lymphoblastoid cell line and formalin-fixed, paraffinembedded tumour tissue [1]. Our Hi-Plex library-building method integrates simple, automated primer design software that enables control of amplicon size. This feature allows complete overlap of read pairs following paired-end sequencing to facilitate stringent downstream filtering of sequencing errors. We recently demonstrated that Hi-Plex using hybrid adapter primers (containing 5′-TruSeq compatible and 3′-Ion Torrent compatible sequences) can produce libraries suitable for both the Ion Torrent (PGM and Proton instruments, Life Technologies, Carlsbad, CA, USA) and TruSeq (MiSeq and HiSeq instruments, Illumina, San Diego, CA, USA) systems, which currently represent the two most commonly used MPS chemistries [2]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call