Abstract
Conventional next generation sequencing analysis has provided important insights into cancer genetics. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. Here, we used the newly developed dual-molecular barcode technology (Ion AmpliSeq HD) to analyze somatic mutations in 24 samples (12 tumor tissues and 12 plasma) from 12 patients with biliary-pancreatic and non-small cell lung cancers. We compared the results between next generation sequencing analysis with or without molecular barcode technologies. The variant allele fraction (VAF) between non-molecular barcode and molecular barcode sequencing was correlated in plasma DNA (R2 = 0.956) and tumor (R2 = 0.935). Both methods successfully detected high VAF mutations, however, rare variants were only identified by molecular barcode sequencing and not by non-molecular barcode sequencing. Some of these rare variants in tumors were annotated as pathogenic, and therefore subclonal driver mutations could be observed. Furthermore, the very low VAF down to 0.17% were identified in cell free DNA in plasma. These results demonstrate that the dual molecular barcode sequencing technologies can sensitively detect rare somatic mutations, and will be important in the investigation of the clonal and subclonal architectures of tumor heterogeneity.
Highlights
Conventional generation sequencing analysis has provided important insights into cancer genetics
We evaluated the performance of Non-molecular barcode (MB) and MB sequencing for detecting somatic mutations in tumors and plasma Cell free DNA (cfDNA) by comparing the MB sequencing data using IonAmpliSeq HD with the results obtained by Non-MB sequencing
The MB sequencing identifies rare variants and shows benefits for analyzing tumor heterogeneity. These results suggested that MB sequencing can be applied for detecting low levels of mutated alleles in the presence of high amounts of wild-type allele
Summary
Conventional generation sequencing analysis has provided important insights into cancer genetics. The very low VAF down to 0.17% were identified in cell free DNA in plasma These results demonstrate that the dual molecular barcode sequencing technologies can sensitively detect rare somatic mutations, and will be important in the investigation of the clonal and subclonal architectures of tumor heterogeneity. Novel assays have been developed to detect rare variants using NGS technologies combined with molecular barcode (MB) technologies[8] This technology is commonly based on the approach that tagging individual DNA fragment with short random oligonucleotides called as unique molecular identifiers (UMI) or unique molecular tag (UMT)[8,9,10]. The molecular barcode discriminates original DNA, clusters the identical barcoded-reads and excludes error-prone nucleotide changes Based on these technologies, Kinde et al developed the approach of massively parallel sequencing using MB, which is called as Safe-Sequencing System (Safe-SeqS)[8]. An alternative amplicon-based method called IonAmpliSeq HD was recently launched Thermo Fisher Scientific
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