Novaseq 6000

In order to amplify and sequence the bound polynucleotides, A RAVE based assay was performed. A poly (A) tail was introduced at the 3′end of the polynucleotides using TdT tailing reaction. 20 μl solutions containing dATP, 10 mM Tris–HCl, 50 mM KCl and 1.5 mM MgCl₂ buffer, the extracted bound polynucleotide sequences (10 μl) and TdT 1–2 U/μl were prepared and incubated for 0.5–2 h followed by termination of the reaction by heating the solution to 75°C for 10 min. The resultant product was used directly as the template in PCR. 20 μl solutions containing 1× master mix (10 μl), 0.1–0.5 μM (1 μl) of the forward and reverse primers and 1–5 μl of the DNA sequences with poly (A) template were prepared. PCR reactions were performed over 30–40 cycles consisting of a denaturing step at 94°C for 30 s, an annealing step at 45–65°C for 30 s and an extension step of 72°C for 30 s. The resultant dsDNA libraries were sequenced using a NovaSeq 6000 (Macrogen, South Korea). All sample libraries were prepared using an Illumina TruSeq DNA PCR free library construction (Insert 350 bp) prior to sequencing with a pairwise read.

Genomic DNA extracted from both the tumorous and non-tumorous portions of each patient FFPE specimen was subjected to whole exome sequencing (WES). WES was outsourced to Macrogen Japan Corp and the Novaseq 6000 was used for the WES. The bioinformatics procedures (Figure S1A) were performed as described previously (Ito et al., 2021 (link)). Briefly, after the raw reads were trimmed, the paired reads were mapped onto hg38 references with BWA-MEM (Liu et al., 2013 (link)). The variant calling was performed with Mutect2 in the Genome Analysis Toolkit 4.1 (McKenna et al., 2010 (link)) and the normal panel consisted of 11 non-cancerous tissue data. The filtering conditions were as follows: false discovery rate = 0.01; unique alt read count = 20; min allele fraction = 0.1; and minimum depth = 100. The annotations for the called variants were performed with Annovar (Wang et al., 2010 (link)) with a custom-made 4.7 KJPN variant dataset and COSMIC 90 (Tate et al., 2019 (link)). To do so, we converted hg38 to hg19 with liftOver software (Hinrichs et al., 2006 (link)).