Translational Genomics Research Institute, Phoenix, AZ 85004, USA.
Phoenix Children's Hospital, Phoenix, AZ 85016, USA.
Sci Transl Med. 2021 Feb 17;13(581). doi: 10.1126/scitranslmed.aaz3088.
Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented. Whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we characterized fragmentation patterns in urine cfDNA using whole-genome sequencing. Size distribution of urine cfDNA fragments showed multiple strong peaks between 40 and 120 base pairs (bp) with a modal size of 81- and sharp 10-bp periodicity, suggesting transient protection from complete degradation. These properties were robust to preanalytical perturbations, such as at-home collection and delay in processing. Genome-wide sequencing coverage of urine cfDNA fragments revealed recurrently protected regions (RPRs) conserved across individuals, with partial overlap with nucleosome positioning maps inferred from plasma cfDNA. The ends of cfDNA fragments clustered upstream and downstream of RPRs, and nucleotide frequencies of fragment ends indicated enzymatic digestion of urine cfDNA. Compared to plasma, fragmentation patterns in urine cfDNA showed greater correlation with gene expression and chromatin accessibility in epithelial cells of the urinary tract. We determined that tumor-derived urine cfDNA exhibits a higher frequency of aberrant fragments that end within RPRs. By comparing the fraction of aberrant fragments and nucleotide frequencies of fragment ends, we identified urine samples from cancer patients with an area under the curve of 0.89. Our results revealed nonrandom genomic positioning of urine cfDNA fragments and suggested that analysis of fragmentation patterns across recurrently protected genomic loci may serve as a cancer diagnostic.
尿液中的无细胞 DNA（cfDNA）是一种很有前途的非侵入性诊断分析物。然而，尿液 cfDNA 高度碎片化。这些片段的特征是否反映潜在的基因组结构尚不清楚。在这里，我们使用全基因组测序来描述尿液 cfDNA 中的片段化模式。尿液 cfDNA 片段的大小分布显示出多个在 40 到 120 个碱基对（bp）之间的强峰，模态大小为 81- 和 10-bp 的尖锐周期性，表明其免受完全降解的短暂保护。这些特性对预处理干扰具有稳健性，例如家庭采集和处理延迟。尿液 cfDNA 片段的全基因组测序覆盖揭示了个体间保守的反复保护区域（RPR），与从血浆 cfDNA 推断的核小体定位图谱部分重叠。cfDNA 片段的末端在 RPR 的上下游聚集，并且片段末端的核苷酸频率表明尿液 cfDNA 的酶消化。与血浆相比，尿液 cfDNA 的片段化模式与尿路上皮细胞中的基因表达和染色质可及性的相关性更高。我们确定，肿瘤来源的尿液 cfDNA 表现出更高频率的异常片段，这些片段在 RPR 内结束。通过比较异常片段的分数和片段末端的核苷酸频率，我们鉴定出癌症患者尿液样本的曲线下面积为 0.89。我们的结果揭示了尿液 cfDNA 片段的非随机基因组定位，并表明分析反复保护的基因组位点的片段化模式可能作为癌症诊断。
