# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 2 Materials and Methods  
Existing genomic and transcriptomic reads were mapped to the published _F. radians_ genomic assembly [12] using bowtie2 2.3.0 [20] for 454 and Illumina reads, and BLASR [21] for PacBio reads. All transcriptomic libraries produced from strain Ax BK280 were pooled together for this work. Bowtie mapping was performed on default settings, except for insert sizes in mate-pair and paired-end libraries: 2-5 Kbp for library 14092013, 200-800 bp for library 27022013, and 250-700 bp for library A280-Illumina. BLASR mapping was performed on default settings.  
165 rRNA reads were extracted from all libraries by aligning a whole library to SILVA v138 reference rRNA alignment in mothur 1.41 [22]. All reads that successfully aligned were classified using mothur's naive Bayesian classifier. To visualize the distribution of these reads along the 165 rRNA gene, a proportion of non-gap characters in the alignment of reads to the reference alignment, normalized between libraries, was plotted along the length of this reference. All statistical analyses and visualizations were performed in Python 3.6 using Biopython and Pyplot.  
Diatom cultures of strain AxBK280 were grown in DM medium (V 600 mL) in a incubator at 8-10 \({}^{\circ}\)C and 16 \(\upmu\)mol/m\({}^{2}\cdot\)s light intensity with 12:12 day:night cycle. For DNA isolation, the cells were grown up to a density of 10\({}^{4}\) cells/mL. For the 165 rRNA sequencing, DNA was isolated from diatom cultures according to the protocol available at [[[http://dx.doi.org/10.17504/protocols.io.qh6d9e](http://dx.doi.org/10.17504/protocols.io.qh6d9e)]([http://dx.doi.org/10.17504/protocols.io.qh6d9e](http://dx.doi.org/10.17504/protocols.io.qh6d9e)), last accessed 27 October 2021]. The V4 variable region of the 16S rRNA gene was amplified using universal bacterial primers (F515 5'-GTGCCACAGCMGCCCGGTAA-3' and R806 5'-GGACTACVSSGGTATCTAAT-3') and sequenced on Illumina MiSeq. PCR amplification and sequencing were performed at the Core Centrum \"Genomic Technologies, Proteomics and Cell Biology\" in ARRIAM (All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russia). Samples from 2020 and 2021 were extracted from fresh culture sedimented by centrifugation at 2500\(\times\)_g_, +4 \({}^{\circ}\)C. DNA from 2017 was extracted in 2020 from frozen culture samples, which were sedimented similarly and stored at \(-\)70 \({}^{\circ}\)C.  
Amplicon libraries were analyzed in mothur 1.41 with SILVA v138.1 reference alignment downloaded from mothur's website. The completeness of _Splingomonas_ genome in _F. radians_ genomic assembly was estimated using BUSCO v5 [23] on gVolante web server [24].  
For the phylogenetic analysis of _Splingomonas_ 16S rRNA we have extracted 16S gene from the genomic assembly and constructed 90% majority rule consensus sequences for all amplicon OTUs classified as _Splingomonas_. We have also included the sequence of _F. radians_-associated _Splingomonas_ sp. described in [25]. All representatives of genera _Splingomonas_ and _Novosphingobium_ were extracted from SILVA v138.1; 200 were selected for the analysis using DJ sampling method [26] with Kimura distance. Genus _Novosphingobium_ was included in this analysis because its member was described in [25] as a closest BLAST hit for the sequence of _Splingomonas_ sp. All _F. radians_-associated sequences were aligned to a subset of SILVA alignment using Clustal Omega [27]. Alignment was further corrected manually. ML phylogenetic tree was built using IQtree [28] on both complete alignment and its subset trimmed by amplicon boundaries.