Fig. 1: A schematic Cas9 targeted enrichment and Nano-Pal pipeline for mobile elements using nanopore sequencing.

a Purified genomic DNA (gDNA) is isolated by salting out and then extensively dephosphorylated. Dephosphorylated gDNA is incubated with the Cas9 ribonucleoprotein which is targeted to MEI subfamily-specific sequences near the 3′ end of the element. Taq polymerase (not shown), and dATPs (not shown) monoadenylate DNA ends. b Cas9 cleaved sites are ligated with Oxford Nanopore Technologies (ONT) sequencing adapters and sequenced on a flow cell. Sequencing is bi-directional from the cleavage site. c Nano-Pal scans the nanopore sequencing reads (black bars) after Cas9 enrichment for MEI signal on one or both ends. The yellow bar represents MEI consensus sequence or MEI signals in pairwise comparison of Nano-Pal. d All reads with or without annotated MEI signal are imported into the downstream pipeline. Alignment, classification, and clustering processes are sequentially conducted. Nano-Pal identifies reference and non-reference MEIs followed by the inspection of nanopore-specific non-reference MEIs (see “Methods” section). e Examples illustrating capture and alignment of reads containing non-reference L1Hs signal (top) and reference L1Hs signal (bottom). Aligned reads display a non-reference insertion (top) with L1Hs signal (yellow bar) and flanking genomic sequence (black bar). MEI components of reads in non-reference insertions are displayed as overlapping (soft clipping) due to lack of reference genome MEI annotation (gray bar). Aligned reads display annotated reference L1Hs (bottom, yellow bar), flanked by surrounding genomic sequence (black bar), separated by the Cas9 cleavage site (red triangle). PALMER and RepeatMasker tracks are illustrated in red and blue, respectively.