Cell-Free DNA Blood Collection Tubes Crosslinking Cellular DNA Impeding Nanopore Long-Read Sequencing.

Long-read DNA and RNA sequencing facilitate genome assembly, haplotyping, complex variant calling, and gene isoform identification. Structural variant calling is integral to the molecular characterization of tumors; thus, long-read nanopore DNA sequencing technology is becoming routinely used in cancer research. As a standard practice, high molecular weight (HMW) DNA is extracted from both tumor and matched normal samples from blood or buffy coat to redact germline variants from somatic mutations. However, we found that buffy coat DNA consistently underperformed compared to DNA extracted from tumor tissue. Furthermore, this observation was unique to DNA extracted from buffy coat cells collected in Streck, but not ethylenediaminetetraacetic acid (EDTA), tubes. We therefore investigated whether the released formaldehyde in Streck tubes resulted in DNA crosslinking, which would explain the low data throughput. Indeed, a decrosslinking step during Streck DNA extraction significantly improved data yield and fragment length without compromising data quality. We therefore recommend a tailored DNA extraction protocol of Streck- derived buffy coat samples for nanopore sequencing.