Abstract
BackgroundRecently, a next-generation sequencing (NGS)-based method has been used for the successful detection of circulating tumor DNA (ctDNA) in various cancer types. Thus, the use of NGS on liquid biopsies will improve cancer diagnosis and prognosis. However, the low-allelic fraction of ctDNA poses a challenge for the sensitive and specific detection of tumor variants in cell-free DNA (cfDNA). To distinguish true variants from false positives, the characteristics of errors that occur during sample preparation and sequencing need to be elucidated.MethodsWe generated capture-based targeted deep sequencing data from plasma cfDNA and peripheral blood leucocyte (PBL) gDNA to profile background errors. To reveal cfDNA-associated DNA lesions, background error profiles from two sample types were compared in each nucleotide substitution class.ResultsIn this study, we determined the prevalence of single nucleotide substitutions in cfDNA sequencing data to identify DNA damage preferentially associated with cfDNA. On comparing sequencing errors between cfDNA and cellular genomic DNA (gDNA), we observed that the total substitution error rates in cfDNA were significantly higher than those in gDNA. When the substitution errors were divided into 12 substitution error classes, C:G>T:A substitution errors constituted the largest difference between cfDNA and gDNA samples. When the substitution error rates were estimated based on the location of DNA-fragment substitutions, the differences in error rates of most substitution classes between cfDNA and gDNA samples were observed only at the ends of the DNA fragments. In contrast, C:G>T:A substitution errors in the cfDNA samples were not particularly associated with DNA-fragment ends. All observations were verified in an independent dataset.ConclusionsOur data suggested that cytosine deamination increased in cfDNA compared to that in cellular gDNA. Such an observation might be due to the attenuation of DNA damage repair before the release of cfDNA and/or the accumulation of cytosine deamination after it. These findings can contribute to a better understanding of cfDNA-associated DNA damage, which will enable the accurate analysis of somatic variants present in cfDNA at an extremely low frequency.
Highlights
A next-generation sequencing (NGS)-based method has been used for the successful detec‐ tion of circulating tumor DNA in various cancer types
Increased C:G>T:A substitution errors in plasma cell-free DNA (cfDNA) We assumed that the increase in DNA damage in cfDNA compared to that in cellular genomic DNA would result in increased errors in cfDNA sequencing data compared to gDNA data
Our data strongly suggest that the increase in C:G > T:A errors in the cfDNA samples was not due to technical artifacts but rather due to sample type-associated attributes. These results demonstrated that C:G > T:A substitution errors increased in cfDNA compared to cellular gDNA, primarily leading to an overall increase in the error rate for cfDNA
Summary
A next-generation sequencing (NGS)-based method has been used for the successful detec‐ tion of circulating tumor DNA (ctDNA) in various cancer types. The low-allelic fraction of ctDNA poses a challenge for the sensitive and specific detection of tumor variants in cell-free DNA (cfDNA). Several challenges remain with respect to the clinical application of cancer diagnosis and monitoring through cfDNA analysis by targeted deep sequencing [5, 8]. The most critical technical challenge is posed by the low allelic frequency of tumor-derived cfDNA fragments, which is often far below 1% [9, 10]. To detect such low-frequency variants, the detection method has to be extremely sensitive and extremely specific. A depth of unique coverage of approximately 500 × is usually reported to be sufficient to profile genetic alterations in tumor specimens [12], but many cfDNA sequencing studies have aimed for a higher depth of unique coverage of 2000–10,000 × [13, 14]
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