Detection of somatic mutation with high sensitivity using Next-generation sequencing


The high sensitivity is required to detect mutation from cell-free DNA whose ratio is less than 1%. We conducted variant analyses of EGFR L861Q, del E746 -A750, L858R and T790M, which are all well-known variation in lung tumor. The samples we used in this study was"EGFR Quantitative Multiplex DNA Reference Standard" made by Horizon Diagnostics. Mutation rates of these EGFR were set as 12.5%, 4.1%, 1.4% and 0.0% (wild type) respectively.


illumina TruSight Tumor panel and Miseq platform were used for sequencing process. Software "GATK" and "MiSeq Reporter Amplicon - DS Workflow" were used to detect four variants. GATK identified L861Q with 12.5%, although failed to detect another three mutations with all rates.

DS Workflow could identify del E746 - A750, L858R and T790M with 12.5% and 4.1% respectively, but could not detect L861Q with 4.1%, nor all mutation with 1.4%. From the results, we refined our protocol with the following two aspects.


1) Quantitative reference standard sample in which mutation rate was verified in advance should be used to identify variants with high sensitivity.

2) The variant calling software tuned for somatic mutation should be used to identify variants with super low rate.


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