Gm12878 cell line

K562 (Chronic Myelogenous Leukemia/Erythro Leukemia) cell line was obtained from NCCS Pune (India) and cultured in RPMI medium (Gibco), Hek293T cells were obtained from the American Type Culture Collection (ATCC, CRL-1573) and were cultured in Dulbecco's modified Eagle's medium (Sigma); both were supplemented with 10% fetal bovine serum (Invitrogen); GM12878 cell line was obtained from Coriell Institute, USA and cultured in RPMI 1640 medium with 15% FBS (not heat-inactivated). all were supplemented with 100 units/ml penicillin-streptomycin solution (Sigma) at 37 °C in a humidified chamber with 5% CO₂. All fine chemicals were purchased from Sigma Aldrich and Life Technologies unless otherwise specified.

A genomic DNA (gDNA) standard product (GeneWell Biotechnology Co., Ltd, Shenzhen, Guangdong, China) with a tumor fraction of about 5% was used in this study, and it contained 7 hotspot mutations, BRAF-p.V600E (8.00%), EGFR-p.L858R (5.00%), EGFR-p.T790M (5.00%), KRAS-p.G12C (5.00%), KRAS-p.G13D (5.00%), EGFR-p.G719A (5.00%) and EGFR-p. E746A750del (5.00%). The original gDNA standard product, its 5-diluted and 10-diluted samples with the GM12878 cell line (Coriell Institute, Camden, New Jersey, USA) and the GM12878 cell line gDNA samples have also three replicates. We designed 6 forward and reverse amplification primers for these 7 hotspot mutations, which were 5′-CAGCTTGCTGCAATGCACACAAGTT-TTCTGTAGATTTCGAGGCCAGAGTCCTT-3′, 5′-AGTTGGGCTCAGCAAGGTAGGCATC-TGATTCCAATGCCATCCACTTGATAGG-3′, 5′-GCCTGACTCAGTGCAGCATGGATTTC-GAGAGATGACGGGCAACGGCGTAT-3′, 5′-TGCTTGGGATGGAAGTTCTACTC-CATATTGACTTCTAACACTTAGAGGTGG-3′, 5′-TGGTGACATGTTGGTACATCCATCCG-GCCTGAGGTTCAGAGCCATGGACC-3′ and 5′-TGCGTTCGGCACGGTGTATAAGGTA-TCGATTCTGCTTCCCTAGTCCGCTG-3′, respectively. Then we performed PCR amplification, end repaired, and ligated nanopore sequencing adapters to build sequencing libraries. Finally, the nanopore sequencing was performed on the QNome-9604 instrument according to the manufacturer’s instructions (Qitan Technology (Beijing) Co., Ltd, Beijing, China), which is a new nanopore sequencing platform.