P42
Nanopore and Illumina sequencing: two inclusive technologies to interrogate the Mycobacterium tuberculosis genome
F Di Marco(1) A Spitaleri(1,2) S Battaglia(1) A M Cabibbe(1) D M Cirillo(1)
1:San Raffaele Scientific Institute; 2:University Vita-Salute San Raffaele
Background
Nowadays, long-reads sequencing (LRS, e.g. Oxford Nanopore Technologies) is acknowledged as a valid alternative to short-reads sequencing (SRS, e.g. Illumina). Both techniques show limitations overcome by the other one, in such ways that both can be exploited for a third solution, namely Hybrid approach. This includes correcting LRS high-rate error using SRS but enabling adequate coverage of challenging regions through LRS. In this study, we aim to evaluate the SRS/LRS quality and performance on M. tuberculosis (MTB) genome resolution.
Methods
We analyzed two distinct outbreaks of MTB clinical isolates (13 strains) sequenced by Illumina and LRS. The reads generated were combined to produce corrected long-reads using Ratatosk (Hybrid). Coverage breadth was evaluated using mosdepth. Phylogenetic analysis was performed using MTBseq. Samples showing less than 6 SNPs were classified as closely genetically related. Genome assemblies from different approaches were performed using Flye and Unicycler.
Results
Coverage breadth to H37Rv reference genome was significantly different among the approaches (p<0.001): SRS scored lower coverage, particularly at of highly-variable-repetitive regions level. The presence of two clusters was confirmed by all approaches although Hybrid approach highlighted few differences in PE/PPE regions intra-cluster. Considering assembly quality, Hybrid and LRS outperformed SRS (e.g. Number of genes, fraction of retrieved genes and NG50).
Conclusions
SRS and LRS show complementary limitations and strengths. SRS poorly resolves MTB challenging regions whereas SRS overcomes this issue but at the cost of lower quality. Hybrid approach, exploiting both, would enable comprehensive MTB genomics analyses, including evolution and diversity, pathogenicity and drug resistance.