In order to estimate the insert locations, all strains have beed subject to nanopore sequencing using GridION X5 from Oxford Nanopore Technologies. For each strain we assembled a genome and locate the inserts on that genome as will be discussed further bellow.
The nextflow workflow, NanoQCtrim, was used for quality control and trimming of the reads. This workflow uses NanoPlot (v. 1.32.0) for quality control and trims adaptors using downpore (v. 0.3.3). Default parameters were used for Nanoplot. For Downpore, the identity matching threshold for the adaptors found in the middle was increased to 100% (default is 85%). No adapter sequences were identified in the final assemblies based on a minimap2 alignment of adapters using default parameters.
We used Flye (v.2.8.2-b1691) for de novo assembly of each of the strain's genomes. Quality of the assemblies were assessed using the nextflow pipeline assemblyStats. The pipeline provides some useful statistics about the assembly as well as BUSCO score. Busco version of v4.1.4_cv1 with the Fungi library (fungi_odb10) was used to test for the completeness of the genome assembly.
Minimap2 (v. 2.2-r409) was used for all the alignments performed during this study. The known insert sequences were aligned to each assembled genome. Upstream sequence of each aligned insert to the assembled genome was extracted (10,000 bases) and then aligned to the reference genomes (ASM694211v1 and ASM694211v1) to identify the insert location.
In order to visualize pairwise comparison between sequences we used a desktop app (re-DOT-able ) from Babraham Bioinformatics to plot interactively and also save the plots as images. We made pairwise comparisons between the reference genome and each of the seven assembled genomes to look for insert location and for any genome re-arrangements. In order to verify the location, we also plotted each assembly against the relevant insert sequences. For detailed information regarding this app, Simon Andrews, the developer has a really good video tutorial.