AbstractMapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.Author summaryMapping consists in the alignment of reads (i.e., DNA fragments) obtained through high-throughput genome sequencing to a previously assembled reference sequence. It is a common practice in genomic studies to use a single reference for mapping, usually the ‘reference genome’ of a species —a high-quality assembly. However, the selection of an optimal reference is hindered by intrinsic intra-species genetic variability, particularly in bacteria. Biases/errors due to reference choice for mapping in bacteria have been identified. These are mainly originated in alignment errors due to genetic differences between the reference genome and the read sequences. Eventually, they could lead to misidentification of variants and biased reconstruction of phylogenetic trees (which reflect ancestry between different bacterial lineages). However, a systematic work on the effects of reference choice in different bacterial species is still missing, particularly regarding its impact on phylogenies. This work intended to fill that gap. The impact of reference choice has proved to be pervasive in the five bacterial species that we have studied and, in some cases, alterations in phylogenetic trees could lead to incorrect epidemiological inferences. Hence, the use of different reference genomes may be prescriptive to assess the potential biases of mapping.
A Complex of Badnavirus Species Infecting Cacao Reveals Mixed Infections, Extensive Genomic Variability, and Interspecific Recombination