Fig. 1

Library preparation and sequencing workflow for both PacBio and ONT. PacBio workflow: DNA is first extracted from blood or cell lines and then sheared to the desired fragment size (typically at 15–25 kbp). After DNA end repair, fragments are ligated to adapters to form circular molecules called SMRTbells. Each SMRTbell is bound by a polymerase and loaded into a single-molecule, real-time (SMRT) cell. Once the sequencing library is loaded into the SMRT cells, each SMRTbell is immobilized at the bottom of one zero-mode waveguide (ZMW). Next, fluorescently labelled deoxynucleoside triphosphates (dNTPs) are added into the wells and sequencing begins. The polymerase starts incorporating dNTP to the new DNA strand. Each incorporated fluorescent dNTP remains briefly at the bottom of the well, where a light pulse from the bottom excites the fluorophore, which is captured by a camera; the fluorophore is then released after nucleotide incorporation. Erroneous stimulation of unincorporated dNTPs can rarely occur if they are particularly close to the bottom of the ZMWs. These occurrences contribute to the error rate of PacBio sequencing. Since modified bases slightly delay the action of the polymerase, CpG methylation can be identified. *Estimate for sequencing on Revio, which has not been extensively tested. ONT workflow: DNA extraction for ONT sequencing can depend on the desired read lengths and may be either a column-based or other extraction. Quality control steps could include an assay to evaluate contamination from the DNA extraction step and recovered fragment length. For DNA sequencing, libraries are typically prepared using either a rapid transposase-based kit, or a longer ligation-based prep that preserves fragment lengths. Libraries are loaded on the flow cell and run for the desired amount of time, with washes as needed based on flow cell performance. Sequencing data can be base called on the machine or transferred to a remote host for processing