An article published in GenomeMedicine discusses a single-molecule sequencing method that enables the analysis of cell-free DNA methylation. This approach has the potential to impact liquid biopsy diagnostics for cancer detection and characterization.
]. Briefly, 25 μl of extracted cfDNA was diluted with 25 μl of water. The sample DNA underwent end-repair and A-tailing with conditions of 20 °C for 30 min and 65 °C for 30 min . We ligated native barcodes using 5 μl of each barcoded adapter following the standard reaction volumes in the KAPA HyperPrep workflow. We used a thermocycler for the ligation step for 4.5 h incubation at 20 °C before holding at 4 °C overnight to maximize the ligation yield.
After the ligation step, 88 μl of Mag-Bind Total NGS beads were added and mixed to each reaction. After incubation for 5 min, the mixtures were pooled together into a 50-μl centrifuge tube. The beads were magnetized and washed with 80% ethanol using a DynaMag separation rack before eluting in 600 μl of 10 mM Tris–HCl pH 8.0 buffer. We performed a second bead cleanup step with 900 μl Mag-Bind Total NGS beads and the same magnetic rack procedure. The elution solution was 50 μl 10 mM Tris–HCl pH 8.
We improved the preparation of multiplex sequencing libraries. For the Oxford Nanopore platform, multiplexing is restricted to the AMII adapter, which has the same motor protein family as the LSK109 sequencing chemistry. This adapter has a significant disadvantage for short fragment libraries because it incurs active consumption of on-chip “fuel” of idle sequencing molecules, leading to rapid flow cell exhaustion.
We generated sequencing libraries for tumor tissue and PBMCs with 1–2 μg of extracted genomic DNA. For some tissue and buffy coat samples with low extraction yields , we used the entire amount of extracted DNA for library preparation. We followed the standard Kapa HyperPrep library preparation kit protocol using 5 μl of AMX-F adapter without barcoding. Each sample was loaded into its own PromethION flow cell for sequencing.
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