The detection of somatic variants in heterogeneous cell populations is crucial in a wide variety of applications such as evolution, the study of immunological diversity and particularly, in cancer genetics where genetic heterogeneity is believed to be the driver of cancer initiation and progression1. Unlike inherited mutations, somatic variants often occur at very low allele frequencies that require sensitive methods for detection. Next generation sequencing (NGS) now allows for parallel high resolution sequencing of billions of nucleotides that has revolutionized the field of genetics. Although theoretically, subclonal DNA populations of any size should be detectable when deep sequencing sufficient numbers of molecules, the identification of rare variants is practically limited by the error rate associated with the sequencing process2.
HaloPlexHS is a high sensitivity targeted sequencing method based on the HaloPlex next generation PCR technology that incorporates >106 unique molecular barcodes in the DNA library, allowing for the identification of duplicate reads hence significantly improving base calling accuracy even at low allelic frequencies compared to conventional NGS methods. Figure 1 shows the detection of allele frequencies down to 0.5% in HapMap mixtures at fixed ratios.
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Excellent Performance and Accuracy
HaloPlexHS provides high coverage of target bases and on-target specificity plus excellent uniformity in both high quality and FFPE samples, ensuring that variants of interest are detected without having to perform excessive sequencing (Figures 2A and B). In addition to the unique molecular barcoding of each DNA library fragment, superior accuracy is achieved by the targeting of both DNA strands which allows for differentiation of true variants from PCR or formalin-fixation artifacts. Combined with the ability to easily create gene panels of interest through customization using SureDesign and compatibility with leading benchtop sequencing platforms, HaloPlexHS is a flexible and high sensitivity assay well-suited for clinical research.
SureCall 3.0 software is optimized for the analysis of NGS data generated using HaloPlexHS. SureCall performs alignment, de-duplication of reads and variant calling using streamlined, guided workflows (Figure 3). When taken together with an efficient HaloPlexHS workflow which allows for complete target enrichment in less than 6 hours, time to results is reduced.
1. Kennedy, S.R et al (2014) Nature protocol 9(11): 2586-2606 2. Gundry, M and Vijg, J (2012) Mutation R4esearch 729: 1-15
For Research Use Only. Not for use in diagnostic procedures