Fig 2: Workflow for linearizing, pooling, and sequencing plasmids on the PacBio RS II and ONT MinION long-read platforms. Each plasmid was cut with the restriction enzyme identified in the respective plasmid maps (Fig. 1), and at the specified location. After linearizing each plasmid independently, the plasmids were pooled and cleaned. We then sequenced the same pool on the PacBio RS II and Oxford Nanopore Technologies’ (ONT) MinION using their respective library preparation protocols. After sequencing, reads from each plasmid were identified using BLAST and then aligned to their respective reference sequences using graphmap, as preparation for downstream comparisons

Fig 3: Workflow for hybridisation and sequencing of long-fragment-enriched pathogen DNA. (a) Enrichment and library preparation for the Oxford Nanopore MinION™ sequencer. (b) non-enriched Nanopore libraries, (c) library preparation for long-fragment-enriched Illumina control experiments.

Fig 4: Comparison of CpG methylation statuses of ecDNA and its native chromosomal locus showed hypomethylation of gene promoters on ecDNA.a, Isolation of ecDNA (guide A) and the corresponding chromosomal locus (guides E + F) from GBM39 neurospheres by CRISPR-CATCH followed by detection of 5mC-CpG methylation by nanopore sequencing. b, Negative correlation between mean methylation probabilities of ATAC-seq peaks and their ATAC-seq signals (Pearson’s R, two-sided test; error bands represent 95% confidence intervals). c, Aggregated levels of relative CpG methylation of ecDNA compared to the chromosomal locus at top 50 ATAC-seq peaks in the ecDNA-amplified region. Mean methylation frequencies were calculated in 100-bp windows sliding every 10 bp. Relative frequencies were quantified from standardized residuals for a linear regression model for mean frequencies on ecDNA vs. chromosomal DNA (Methods). d, Bulk ATAC-seq track with differentially methylated regions annotated (Methods; two-sided z-test, P values were Benjamini-Hochberg adjusted; regions with P < 0.005 were considered significant). e, Relative CpG methylation of ecDNA compared to the chromosomal locus in differential regions and concordance with accessibility by ATAC-seq and nucleosome positioning by MNase-seq. Mean methylation frequencies were calculated in 100-bp windows sliding every 10 bp. Relative frequencies were quantified from standardized residuals for a linear regression model for mean frequencies on ecDNA vs. chromosomal DNA (Methods). f, From top to bottom: Loess-smoothed methylation probability around the EGFR promoter (error band represents 95% confidence intervals); nanopore sequencing reads showing CpG methylation calls (gray denotes regions with no CpG sites); heatmap showing co-occurrence probabilities of unmethylated CpG sites on the same molecules; heatmap showing co-occurrence probabilities of methylated CpG sites on the same molecules (Methods). Reads were collected using a MinION sequencer (Oxford Nanopore Technologies).

Fig 5: Sample and data collection scheme. The figure shows the cross of a single male and female mosquito to produce female F1 offspring. The F0 father and mother were sequenced individually using Illumina HiSeq to obtain short reads. Genomic DNA from a pool of the female F1 sibling was sequenced using three Oxford Nanopore MinION flow cells to produce long reads. Genomic DNA from a pool of the paternal half-sibling F1 males was used to generate Bionano DLS optical mapping. Oxford Nanopore reads were used to generate the contigs using Canu (Koren et al., 2017) and the Canu contigs were polished using the parental Illumina reads. The polished Canu contigs were scaffolded using the Bionano DLS optical map to generate the AalbS3 assembly.