Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing

Ribosomal DNA (rDNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large scale exploration of environmental microbial diversity through metabarcoding approaches have been focused mainly on the hypervariable regions V4 and V9 of the 18S rDNA gene. Yet, the accurate interpretation of such environmental surveys is hampered by technical (e.g., PCR and sequencing errors) and biological biases (e.g., intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with two different high-throughput sequencing platforms: Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet in some Spumellaria specimens we found two different copies of the V4 with a similarity lower than 97%. From the different sequencing methods, Illumina showed the highest number of contaminations (i.e., environmental DNA, cross-contamination, tag-jumping), revealed by its high sequencing depth; and Minion showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results, highlight the requirement for a careful interpretation of Illumina based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full environmental rDNA metabarcoding surveys.<br> <br> Here you can find 7 files gathering raw data obtained in this study and its associated metadata: <br> Data obtained by <strong>Sanger</strong> sequencing:<br> sanger_raw.fasta.gz: A fasta file containing the sequences with the following sequence names: "Cell-identifier_primer_barcode_well"; please note the underscore delimiting fields. <br> Data obtained by <strong>MinION (ONT)</strong> sequencing: minion_raw.fastq.gz: Fastq file. minion_raw.fast5.tar.gz: Fast5 native MinION raw data for future implementations or re-analysis. info_minion_demultiplex.txt: The information file for demultiplexing MinION data. <br> Data obtained by <strong>Illumina</strong> sequencing: R1.fastq.gz: R1 fastq file. R2.fastq.gz: R2 fastq file. info_illumina_tags_cutadapt.txt: The information file for demultiplexing Illumina data.<br> <br> For further details or if using these datasets, please see/cite the following study:<br> Sandin MM, Romac S, Not F (2022) <strong>Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing</strong>. <em>in press, Environmental Microbiology</em>; <strong>https://doi.org/10.1111/1462-2920.16081</strong><br>