Sequencing, Alignment, and Gene-Expression Quantification.

All single-cell libraries were sequenced with a customized paired end with dual indexing (98/14/8/10-bp) format according to the recommendation by 10X Genomics. All single-cell libraries were sequenced on a MiSeq system (Illumina) or a NextSeq 500 system (Illumina) using the MiSeq Reagent v3 Kit (Illumina) or the NextSeq 500 High Output v2 Kit (Illumina), respectively. The data were aligned and quantified using the Cell Ranger Single-Cell Software Suite (version 1.0) as described by Zheng et al. (28). In short, samples were demultiplexed based on the 8-bp sample index, 10-bp UMI tags, and the 14-bp GemCode barcode. The 98-bp-long read 1 containing the cDNA sequence was aligned using STAR (68) against the hg19 human reference genome. UMI quantification, GemCode, and cell barcodes filtering based on error detection by Hamming distance were performed as described by Zheng et al. (28).

For alignment of the plasma RNA library, adaptor sequences and low-quality bases on the fragment ends (i.e., quality score < 5) were trimmed, and reads were aligned to the human reference genome (hg19) using the TopHat (v2.0.4) software with the following parameters: transcriptome mismatches = 3; mate-std-dev = 50; genome-read-mismatches = 3 with the pair end alignment option as well as the annotated gene model file downloaded from University of California, Santa Cruz, Genome Browser (genome.ucsc.edu/). Gene-expression quantification was performed by an in-house script quantifying the number of reads overlapping with exonic regions on genes annotated in the Ensembl GTFs (GRCh37.p13).