Fig 1.
Construction of the sequencing library.
a) Cell-free DNA was end-repaired, phosphorylated, and ligated to adapters. DNA fragments with adapters were subjected to linear amplification using a region-specific primer mix. To construct sequencing libraries, the products were amplified using primers including sequences indispensable for the Ion Torrent sequencing system. b) Schematic depicting primer design. To cover the entire intron 19 of ALK, we designed a set of 42 tiling primers.
Fig 2.
Flowchart of sequencing data analysis.
Black and gray arrows denote fusion detection and re-quantification processes, respectively.
Fig 3.
Estimating allele fraction of an artificial fusion by NGS assay.
Normal and EML4-ALK reference standard DNAs were mixed over a range of fusion allele ratios and used as templates for NGS assays. Experiments were performed four times for 0.5, 1, and 5% allele ratios, twice for 10%, and once for 20% ratios. Circles and crosses indicate fusion allele fractions before or after re-quantification of sequencing reads by alignment using 20 bp breakpoint sequences, respectively.
Fig 4.
Comparison of fusion allele fractions between NGS assays and qPCR.
After re-aligning NGS reads to each breakpoint sequence, quantification of fusion allele fractions using NGS and qPCR were closely correlated (crosses) as opposed to estimates without re-quantification (circles). Data from samples, AK03, AK04, AK07, AK09, and AK10 are plotted. AK03 and AK04 were assayed twice (see Table 1).
Table 1.
Breakpoints of fusion alleles detected from ALK-positive NSCLC patients.
Fig 5.
Graphic representation of ALK fusion breakpoints.
a) ALK intron 19, b) EML4 intron 6, c) intron 13, and d) intron 20 are shown. Regions where breakpoints are absent are shaded pink. Breakpoints of fusions are denoted by black (reported in previous studies), blue (detected in present study), and white circles (reported in ALCL study). Colored lines indicate repetitive elements, Alu (red), hAT-Charlie DNA transposon (green), L1/L2 (blue), AT-rich (brown), MIR (Mammalian-wide Interspersed Repeat) (orange), simple repeat (black), TcMar-Tigger DNA transposon (light green), and LTR (Long Terminal Repeat) (purple).