Extracellular RNAs (exRNAs) are present in human serum. It remains unclear to what extent these circulating exRNAs may reflect human physiologic and disease states. Here, we developed SILVER-seq (Small Input Liquid Volume Extracellular RNA Sequencing) to efficiently sequence both integral and fragmented exRNAs from a small droplet (5 μL to 7 μL) of liquid biopsy. We calibrated SILVER-seq in reference to other RNA sequencing methods based on milliliters of input serum and quantified droplet-to-droplet and donor-to-donor variations. We carried out SILVER-seq on more than 150 serum droplets from male and female donors ranging from 18 y to 48 y of age. SILVER-seq detected exRNAs from more than a quarter of the human genes, including small RNAs and fragments of mRNAs and long noncoding RNAs (lncRNAs). The detected exRNAs included those derived from genes with tissue (e.g., brain)-specific expression. The exRNA expression levels separated the male and female samples and were correlated with chronological age. Noncancer and breast cancer donors exhibited pronounced differences, whereas donors with or without cancer recurrence exhibited moderate differences in exRNA expression patterns. Even without using differentially expressed exRNAs as features, nearly all cancer and noncancer samples and a large portion of the recurrence and nonrecurrence samples could be correctly classified by exRNA expression values. These data suggest the potential of using exRNAs in a single droplet of serum for liquid biopsy-based diagnostics.

Using a magnetic tweezers (MT) apparatus and an atomic force microscope (AFM), we studied the condensation of DNA induced by the cationic gemini surfactant hexyl-alpha,omega-bisdodecyldimethylammonium bromide (C12 C6 C12 Br2). Stepwise condensation events were found for forces from 0.5 to 4 pN, with a decrease in DNA extension by approximately 100 nm in each condensation event. Applied stretching forces larger than 6 pN were found to be able to decondense the condensates in a similar intermittent stepwise manner, but with approximately 60 nm of DNA released in each decondensation event. These observations are consistent with AFM images that show beadlike structures on DNA. The results lead to a model in which, during condensation, independent beadlike structures are initially formed on DNA, and as the local density of the beads increases, they compact into higher-order structures while maintaining their independence. The condensation process is different from any previously reported condensation process, but it is somewhat similar to that of chromatin assembly.

Summary Circulating cell-free DNA (cfDNA) is a promising biomarker for the diagnosis and prognosis of many diseases, including cancer. The genome-wide non-random fragmentation patterns of cfDNA are associated with the nucleosomal protection, epigenetic environment, and gene expression in the cell types that contributed to cfDNA. However, current progress on the development of computational methods and understanding of molecular mechanisms behind cfDNA fragmentation patterns is significantly limited by the controlled-access of cfDNA whole-genome sequencing (WGS) dataset. Here, we present FinaleDB (FragmentatIoN AnaLysis of cEll-free DNA DataBase), a comprehensive database to host thousands of uniformly processed and curated de-identified cfDNA WGS datasets across different pathological conditions. Furthermore, FinaleDB comes with a fragmentation genome browser, from which users can seamlessly integrate thousands of other omics data in different cell types to experience a comprehensive view of both gene-regulatory landscape and cfDNA fragmentation patterns. Availability and implementation FinaleDB service: http://finaledb.research.cchmc.org/. FinaleDB source code: https://github.com/epifluidlab/finaledb_portal, https://github.com/epifluidlab/finaledb_workflow Supplementary information Supplementary data are available at Bioinformatics online.