PMC9675175_01
Genomic evidence for 

Y. pseudotuberculosis

 at the sequencing and genomic assembly level. (a) Graphical representation of the two-pronged approach for genomic identification of 

Y. pseudotuberculosis

. Software utilised in this workflow are listed in parenthesis. (b) Kraken2 classification report as a Sankey plot showing the top three members in each taxonomical level. The taxonomical level on the x-axis are Domain, Phylum, Family, Genus and Species. Figure was generated using Pavian version 1.0. (c) Minimum spanning tree generated from 321 out of 500 loci from the 

Yersinia

 cgMLST scheme that were present in all complete genomes in our dataset (Supplementary File 2). Each genome is represented as a node and connecting lines represent number of allelic differences, scaled according to the scale bar. Image was generated using GrapeTree.
acmi-4-422-g001.jpg
PMC9675175_01_acmi-4-422-g001.jpg
F1
Nanopore sequencing on a MinION (Oxford Nanopore Technologies) was performed for 20 h with a 'two-pronged' approach to gather multiple lines of evidence at the reads and assembly level (Fig. 1a; Supplementary File 1, available in the online version of this article). Of the 336 622 trimmed reads, 81.7 % were classified as   Y. pseudotuberculosis   as opposed to 1.74 % as   Y. pestis   (Fig. 1b). In addition, core genome MLST (cgMLST) showed that this isolate clustered together with other   Y. pseudotuberculosis   genomes (Fig. 1c, Supplementary File 2).
Retrospective analysis of generated FAST5 files. (a) Stacked histogram showing proportion of reads in each time point classified as either Y. pseudotuberculosis, Y. pestis or other. Total number of reads for each time point is listed on top of each histogram. Image generated using ggplot2. (b) Graphical representation of polished long-read assembly status and MLST result of the six time points used in the retrospective analysis. Image generated using Bandage version 0.81. (c) Instances of the 144 identified 'SNPs' between the assembly generated from 10 h of sequencing (top) and the initial assembly (middle) with positions scattered across the entire assembly (bottom). Image generated using snipit (https://github.com/aineniamh/snipit).
acmi-4-422-g002.jpg
PMC9675175_01_acmi-4-422-g002.jpg
F2
In addition, core genome MLST (cgMLST) showed that this isolate clustered together with other   Y. pseudotuberculosis   genomes (Fig. 1c, Supplementary File 2). Retrospective analysis showed that data generated within 2 h would return the same results on both the read and assembly level. (Fig. 2). A contiguous assembly, albeit 59 bases shorter, could be generated from reads 10 h into sequencing with genomic difference localised to 141 'SNPs' (Fig. 2), 446 insertions and 456 deletions.