Variant with asterisk (*) shows the mutations which are detected by visual inspection of Binary SAM (BAM) files by Ion Reporter Genomic Viewer. Tumor types (CCC, cholangiocarcinoma GBC, gallbladder cancer PC, pancreatic cancer, NSCLC, non-small cell lung cancer) were denoted under the case number. Gray box indicates no identified alterations. Variant allele fraction (%) is shown in each box and is indicated by the graduated color scale from 1% (light blue) to 100% (dark blue). Identical mutations in plasma cfDNA corresponding to mutations in tumor samples were detected. Sequencing was performed with non-molecular barcode (Non-MB) and molecular barcode (MB) technologies. Samples were collected from 12 patients with biliary-pancreatic (Case #1–4) and non-small cell lung cancers (Case #5–12). Heat map shows the mutation profiles in tumors and plasma cfDNA using both sequencing methods.
#Free barcode free
Molecular barcode sequencing sensitively detects the tumor-derived mutations in plasma cell free DNA (cfDNA).
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. Furthermore, the very low VAF down to 0.17% were identified in cell free DNA in plasma.
#Free barcode driver
Some of these rare variants in tumors were annotated as pathogenic, and therefore subclonal driver mutations could be observed. Both methods successfully detected high VAF mutations, however, rare variants were only identified by molecular barcode sequencing and not by non-molecular barcode sequencing. The variant allele fraction (VAF) between non-molecular barcode and molecular barcode sequencing was correlated in plasma DNA (R 2 = 0.956) and tumor (R 2 = 0.935). We compared the results between next generation sequencing analysis with or without molecular barcode technologies. 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. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. Conventional next generation sequencing analysis has provided important insights into cancer genetics.