# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 3 Results  
The list of genes and scaffolds thought to contain _Splingomonas_ contamination was generously provided by [PERSON]. In total, this list includes 3644 genes on 697 scaffolds with total length of approx. 4 Mbp. Their results confirm high sequence identity and similar GC-content between these scaffolds and genomes of various Sphingomonadales, but that does not automatically imply contamination. Although a huge wave of HGTs from _Splingomonas_ to _F. radians_ is unlikely, it is not outright impossible. To validate that these scaffolds are not, in fact, valid _F. radians_ genes, we have mapped available transcriptomic reads on the whole genome assembly. The reasoning behind this is that the cDNA used in transcriptomic project was amplified using oligo(dT) primers that bind polyadenylated eukaryotic mRNA. _Splingomonas_ mRNA, lacking polyadenylation, would thus be underrepresented or even completely absent from transcriptomic dataset. Therefore, if the scaffolds in question are poorly covered by the transcriptomic data, it would serve as further evidence of their bacterial origin. As Figure 1 shows, transcriptomic coverage of the supposedly _Splingomonas_-derived scaffolds is extremely low. Only a few genes show any coverage at all, while the majority are completely absent from transcriptomic data. Absence of these genes from transcriptomic data shows that they are not a part of _F. radians_ active gene repertoire. However, this does not necessarily imply that they are not a part of its genome. There are at least two alternative interpretations: they may be valid _F. radians_ genes not expressed under conditions of our transcriptomic experiment, or they may be pseudogenized. It is also possible that these genes are indeed bacterial, but the contamination was not present in samples used for RNA-seq. According to [16], the scaffolds that contain these genes show remarkable sequence similarity to Sphingomonadales (the authors used maximum likelihood phylogeny to detect origins, and later filtered contigs using BLAST hits to Sphingomonadales with >75% identity and >25% contig coverage) and higher GC-content (63.3% vs. genome average of 42.1%, \(p\) < 2 \(\times\) 10\({}^{-16}\), see Figure 2b in [16] for a more detailed view). Further, no other diatoms were found to contain that many HGT-derived genes acquired from a single source and not shared with at least one other diatom. BUSCO analysis of these scaffolds shows that they include 89.59% of the Sphingomonadales ortholog set (84.58% complete, 5.01% partial). In other words, this nearly complete ortholog set seems to have been sampled from a bacterial genome, which is to be expected if we have sequenced a mixture of _F. radians_ and _Sphingomonas_ sp. cells. Pseudogenization or low expression levels fail to explain these observations, so we can safely assume that genes identified as belonging to _Sphingomonas_ are, in fact, a product of bacterial contamination. To establish whether this contamination persists in all sequencing data, whole-genome sequencing libraries were mapped to the published assembly. The number and proportion of reads mapped to _Sphingomonas_-derived scaffolds are shown in Table 1. For reference, these scaffolds comprise 4 Mbp of the assembly 98.4 Mbp in size, or \(\sim\)4.07% of the total assembly length. Figure 1: The number of transcriptomic reads mapping to putatively _Sphingomonas_-derived and other (putatively _F. radians_-derived) genes. Read counts are given in decimal log scale. These numbers show that reads mapping to the scaffolds in question are overrepresented in 454 libraries from 2011-2012, suggesting that corresponding DNA was present in these samples but not the rest. This is a further argument towards the bacterial origin of these scaffolds and a hint as to when the contamination happened. It is important to note that we considered whole scaffolds to be bacterial, although it is possible that some of them are chimeric, i.e., include fragments from genomes of both _F. radians_ and _Sphingomonas_ sp.