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. Here you can find 7 files gathering raw data obtained in this study and its associated metadata: Data obtained by Sanger sequencing: 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. Data obtained by MinION (ONT) 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. Data obtained by Illumina 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. For further details or if using these datasets, please see/cite the following study: Sandin MM, Romac S, Not F (2022) Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing. in press, Environmental Microbiology; https://doi.org/10.1111/1462-2920.16081

核糖体DNA（rDNA）是真核生物条形码鉴定及不同分类层级系统发育分类的重要标记基因。通过元条形码（metabarcoding）技术大规模探索环境微生物多样性的研究，主要聚焦于18S rDNA基因的高变区V4与V9。然而，此类环境调查的精准解读常受技术偏差（如PCR与测序错误）及生物学偏差（如基因组内变异）的制约。
本研究通过对比桑格测序（Sanger sequencing）与两种不同高通量测序平台——Illumina及牛津纳米孔科技公司（Oxford Nanopore Technologies，MinION）的测序结果，探究了放射虫门（Radiolaria）中罩鞭毛虫类（Nassellaria）与泡沫虫类（Spumellaria）标本的基因组内多样性。分析结果显示，罩鞭毛虫类与泡沫虫类的基因组内变异整体较低，但部分泡沫虫类标本中存在两个相似性低于97%的V4区拷贝。
在不同测序方法中，Illumina因测序深度较高，检出的污染（即环境DNA、交叉污染、标签跳跃）数量最多；而MinION的测序错误率最高（约14%），但其产生的长读长序列（约2900 bp）可用于精准的系统发育重建研究。这些研究结果强调，需谨慎解读基于Illumina的元条形码研究，尤其是针对低丰度扩增子的分析，并为全面开展环境rDNA元条形码调查开辟了未来研究方向。
本研究附带7份文件，包含本研究获得的原始数据及其相关元数据：
桑格测序数据：sanger_raw.fasta.gz：该FASTA文件包含序列，序列命名格式为"Cell-identifier_primer_barcode_well"，请注意字段间以下划线分隔。
MinION（ONT）测序数据：minion_raw.fastq.gz：FASTQ格式文件；minion_raw.fast5.tar.gz：MinION原生FAST5原始数据，供后续研究或重分析使用；info_minion_demultiplex.txt：MinION数据解复用信息文件。
Illumina测序数据：R1.fastq.gz：R1端FASTQ文件；R2.fastq.gz：R2端FASTQ文件；info_illumina_tags_cutadapt.txt：Illumina数据解复用信息文件。
如需进一步了解或使用本数据集，请参阅/引用以下研究：
Sandin MM, Romac S, Not F (2022) Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing. in press, Environmental Microbiology; https://doi.org/10.1111/1462-2920.16081