# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 3 Results
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Library ID, Strain** & **Platform** & **Sequencing Date** & **Total Read Count** &
\begin{tabular}{c} **Number/Proportion of** \\ **Reads Mapped to** \\ **SPHINGOMNAS** \\ **Scaffolds** \\ \end{tabular} \\ \hline GIDRLYB02, G9 & 454 Roche & 4 June 2010 & 587,240 & 10/0.002\% \\ \hline GKAKADZ02, G9 & 454 Roche & 9 July 2010 & 676,802 & 6/0.001\% \\ \hline GW4 JR4A02, G9 & 454 Roche & 3 February 2011 & 319,195 & 6354/1.991\% \\ \hline HHYTHSSC, G9 & 454 Roche & 24 January 2012 & 1,031,775 & 10,376/1.006\% \\ \hline HRUMODM, G9 & 454 Roche & 25 July 2012 & 761,808 & 9575/1.257\% \\ \hline HVPG79F, G9 & 454 Roche & 3 October 2012 & 1,797,450 & 18,942/1.054\% \\ \hline HV19 YS0, G9 & 454 Roche & 10 October 2012 & 1,842,371 & 17,731/0.962\% \\ \hline
27022013, G9 &
\begin{tabular}{c} Illumina MiSeq\({}_{r}\) \\ paired-end & 27 February 2013 & 6,869,020 1 & 2974/0.04\% 1 \\ \hline
14092013, G9 & \begin{tabular}{c} Illumina MiSeq\({}_{r}\) \\ mate pair \\ \end{tabular} & 14 September 2013 & 11,783,953 1 & 2438/0.021\% 1 \\ \hline A280-Illumina, Ax BK280 & \
\begin{tabular}{c} Illumina HiSeq\({}_{r}\) \\ paired-end & 5 August 2016 & 73,655,035 1 & 19,365/0.026\% 1 \\ \hline A280-PB, Ax BK280 &
\begin{tabular}{c} PacBio RS2 \\ \end{tabular} & 2015 & 716,581 & 4088/0.06\% \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Genomic libraries produced from _Fragilaria radians_ strains G9 and Ax BK280. For PE Illumina libraries, the number of pairs is shown instead of the number of reads. Figure 2: Normalized coverage of SILVA v138 reference alignment by bacterial and unclassified reads from various libraries. Libraries 27022013 and 14092013 were pooled for this analysis and shown as “G9-Illumina”. This suspicion is further confirmed by taxonomic analysis of 165 rRNA reads extracted from these libraries. In case of contaminated libraries, it points to _Sphingomonas_, or at least unclassified Sphingomonadales, as a major source of bacterial rRNA. In other libraries, with a single exception, there are no more than a few reads confidently classified as something other than mitochondria or chloroplasts. The exception is library GKAKADZ02; although it appears to be \"clean\" based on a number of reads mapping to _Sphingomonas_ scaffolds, this library contains a large number of 165 rRNA reads derived from diverse bacteria. The origin of these reads can be clarified by plotting the coverage of SILVA reference alignment by non-organellar reads from this library (Figure 2; distribution for library GKAKADZ02 is shown in red). For all other libraries, this plot shows a roughly U-shaped curve with high coverage of alignment ends and a relatively low coverage in the middle. This shape can be explained by the fact that this alignment contains multiple gaps in the 6000-35,000 region including V2-V6 16S rRNA hypervariable regions, so each read is stretched over multiple kilobases of alignment length, producing lower per-base coverage. For library GKAKADZ02, though, the plot suggests that a huge amount of reads covering a very specific region (roughly corresponding to V2-V5 variable regions of rRNA, a segment commonly used in 16S rRNA-based diversity studies) was added to a normal shotgun library. Together with an elevated bacterial diversity this result suggests contamination by an SSU amplicon during storage or sequencing run preparation. Analysis of shotgun libraries has shown that _Sphingomonas_ genomic DNA was only present in the libraries produced from strain G9 during 2011-2012. Later libraries produced from this strain, as well as all libraries produced from strain Ax BK280, appear to be clean. However, we have decided to further validate the purity of Ax BK280 using 16S rRNA amplicon sequencing.