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Background and Aim  The clinical applicability of digital next‐generation sequencing (dNGS), which eliminates polymerase chain reaction (PCR) and sequencing error‐derived noise by using molecular barcodes (MBs), has not been fully evaluated. We evaluated the utility of dNGS of cell‐free DNA (cfDNA) in liquid biopsies obtained from patients with pancreatic cancer.    Methods  Fifty‐eight patients with pancreatic cancer undergoing endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) were included. Samples were subjected to sequencing of 50 cancer‐related genes using next‐generation sequencing (NGS). The results were used as reference gene alterations. NGS of cfDNA from plasma was performed for patients with a mutant allele frequency (MAF) >1% and an absolute mutant number > 10 copies/plasma mL in  KRAS  or  GNAS  by digital PCR. Sequence readings with and without MBs were compared with reference to EUS‐FNA‐derived gene alterations.    Results  The concordance rate between dNGS of cfDNA and EUS‐FNA‐derived gene alterations was higher with than without MBs ( p  = 0.039), and MAF cut‐off values in dNGS could be decreased to 0.2%. dNGS using MBs eliminated PCR and sequencing error by 74% and 68% for  TP53  and all genes, respectively. Overall, dNGS detected mutations in  KRAS  (45%) and  TP53  (26%) and copy number alterations in  CCND2 ,  CCND3 ,  CDK4 ,  FGFR1 , and  MYC , which are targets of molecular‐targeted drugs.    Conclusions  dNGS of cfDNA using MBs is useful for accurate detection of gene alterations even with low levels of MAFs. These results may be used to inform the development of diagnostics and therapeutics that can improve the prognosis of pancreatic cancer.

Digital next‐generation sequencing of cell‐free DNA for pancreatic cancer