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To evaluate any possible DNA contamination in Small Input Liquid Volume Extracellular RNA Sequencing (SILVER-seq), we carried out three types of tests. First, we tested the effectiveness of SILVER-seq’s DNase treatment step. Without DNase treatment, five of the five tested serum samples exhibited detectable cell-free DNA (cfDNA; Fig. 1 A ). After SILVER-seq’s DNase treatment step, none of the serum samples exhibited any detectable cfDNA (Fig. 1 A ), suggesting that SILVER-seq’s DNase treatment step thoroughly digested cfDNA in human serum samples. Fig. 1. DNA contamination tests. ( A ) Concentrations of cfDNA in five human serum samples (IDs: hS01 to hS05, columns) before and after SILVER-seq’s DNase treatment step. Each serum sample (column) was split into two 7-µL aliquots which were subjected to no treatment ( Upper ) or DNase treatment ( Lower ). ND, not detectable. The detection limit of Qubit’s high-sensitivity kit is 0.5 pg/µL. ( B ) Measured concentrations of cfDNA titers (columns) before ( Upper ) and after ( Lower ) SILVER-seq’s DNase treatment step. The detection limit of … [↵][1]2To whom correspondence may be addressed. Email: shuchien{at}ucsd.edu, hisu{at}ucsd.edu, or szhong{at}ucsd.edu. [1]: #xref-corresp-1-1

Significance The SILVER-seq technology enables sequencing extracellular RNAs (exRNAs) from a single droplet of liquid biopsy. This study revealed strong associations between serum exRNA expression levels and the donor’s sex and age. SILVER-seq detected serum exRNAs from the genes that are only expressed in brain, suggesting the possibility of monitoring brain gene expression from a blood test. Classifiers based on exRNA expression levels were able to separate breast cancer patients from control donors. The exRNA-based classifiers could also distinguish the patients with recurrent cancer from other breast cancer patients. The SILVER-seq technology can therefore lead the way to future in vitro diagnostics trials based on finger prick blood, which is more accessible for screening and frequent monitoring of human diseases., 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.

We thank Drs. Hartl and Gao for pointing out the difference in read lengths between the cancer and normal samples (1). Indeed, Illumina discontinued their 50-base pair (bp) sequencing kits after we finished sequencing the cancer samples, forcing us to switch to 75-bp sequencing kits on the normal samples. The different sequencing kits did incur small changes of sequence alignment metrics. While acknowledging the possibility that this may bias the assessment of cancer vs. normal discrimination performance of our method, we believe such a possibility is substantially ameliorated by the following considerations. First, a recent study reported that “with the exception of 25 bp reads, there is little difference for the detection of … [↵][1]1To whom correspondence may be addressed. Email: shuchien{at}ucsd.edu, hisu{at}ucsd.edu, or szhong{at}ucsd.edu. [1]: #xref-corresp-1-1