DNA library construction and sequencing

Urine cfDNA was fragmented to approximately 200-bp size, while germline DNA and tumor DNA were sheared to approximately 250-bp size prior to library preparation using a LE220 Focused Ultrasonicator (Covaris). All urine cfDNA and germline DNA libraries were prepared using the Kapa HyperPrep Kit with custom sequencing adapters containing demultiplexing, deduplicating, and duplex barcodes [21]. Minimum inputs of 32 ng were used for each library preparation reaction. Targeted hybrid-capture was performed per the uCAPP-Seq protocol [28] with the following modifications: For urine cfDNA, 3 or 4 samples were captured with the custom bladder cancer selector panel (S2 and S3 Tables), while 6-plex captures and 12-plex captures were performed for tumor and germline samples, respectively. Libraries were sequenced on an Illumina HiSeq 4000 with 2 × 150 bp paired-end reads. Sequencing quality control metrics including deduplicated depth, on-target rate, fragment size, and duplex recovery rate were reviewed (S4–S6 Tables). We sequenced urine cfDNA from localized bladder cancer patients and healthy donors to a median deduplicated depth of 811×, while germline DNA and tumor DNA were sequenced to a median deduplicated depth of 781× and 1917×, respectively (S4–S6 Tables). Sequences were analyzed for single-nucleotide variants (SNVs) using the uCAPP-seq bioinformatics pipeline with error suppression [20,21]. Briefly, sequencing reads were demultiplexed using sample-level index barcodes, mapped to the reference genome GRCh37/hg19 (February 2009), filtered for properly paired reads, filtered for bases with Phred quality score ≥30, and deduplicated using unique molecular barcodes. The molecular barcoding strategy enabled the identification of duplex-supported reads (S4 and S5 Tables).