T-Cell Clonality Testing by Next Generation Sequencing: A Comparison of Benchtop Sequencing Platforms
Eric Duncavage, Jonathan Schumacher, Philippe Szankasi, Todd Kelley. Washington University in St Louis, St. Louis, MO; ARUP Laboratories, Salt Lake City, UT; University of Utah, Salt Lake CIty, UT
Background: Next-generation sequencing (NGS) is rapidly becoming a standard laboratory technique in molecular onclology, in part due to newer, inexpensive platforms such as the Ion Torrent (Life Technology) and MiSeq (Illumina). One potential application of NGS is the analysis of the T-cell receptor (TCR) gamma repertoire in patients with suspected T-cell malignancies. Current methodologies rely on capillary electrophoresis (CE) of amplified TCR gamma sequences, with clonality determined by peak height over the polyclonal background. This method is not sufficiently sensitive for minimal residual disease testing, and is often confounded by apparent oligoclonal populations. Here we describe a study designed to compare sequencing the TCR gamma repertoire on the Ion Torrent versus the MiSeq.
Design: PCR primers were designed to target TCR V-gamma 2 through V-gamma 11+J gamma rearrangements in a single multiplex reaction. Genomic DNA (400 ng) from six patient samples was subjected to PCR amplification. Amplicons were purified and 500ng indexed for Ion Torrent and MiSeq analysis. Indexed libraries were sequenced in pairs using the Ion Torrent 314 chip or as a single pool on the MiSeq using 2x150bp reads. Contigs were reconstructed from the paired-end MiSeq reads. Data were analyzed by first identifying the most common reads and then mapping reads to a database of TCR sequences. T-cell clonality was established by examining the distribution of TCR sequences.
Results: Data from both the Ion Torrent and MiSeq correctly identified 2/2 cases called polyclonal by CE and 2/2 cases called clonal by CE. In the 2 cases called oligoclonal by CE, both the MiSeq and the Ion Torrent identified the same small clonal populations, as determined by the V region sequence. While the overall results of the MiSeq and Ion Torrent were similar, the Ion Torrent produced a larger number of background sequences, which obscured analysis by PCR product size distribution.
Conclusions: T-cell clonality testing by NGS is a robust alternative to standard CE detection and provides the ability to accurately identify small populations that may be missed by CE. Further, as the exact clonal V region sequence is known, NGS methods have the potential to improve minimal residual disease detection and establish a clonal relationship between recurrent specimens.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 242, Tuesday Morning