Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes

Abstract

Background De novo transcriptome assemblies are required prior to analyzing RNAseq data from a species without an existing reference genome or transcriptome. Despite the prevalence of transcriptomic studies, the e ects of using di erent work ows, or "pipelines", on the resulting assemblies are poorly understood. Here, a pipeline was programmatically automated and used to assemble and annotate raw transcriptomic short read data collected by the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP). The resulting transcriptome assemblies were evaluated and compared against assemblies that were previously generated with a di erent pipeline developed by the National Center for Genome Research (NCGR). Results New transcriptome assemblies contained the majority of previous contigs as well as new content. On average, 7.8% of the annotated contigs in the new assemblies were novel gene names not found in the previous assemblies. Taxonomic trends were observed in the assembly metrics, with assemblies from the Dino agellata and Ciliophora phyla showing a higher percentage of open reading frames and number of contigs than transcriptomes from other phyla. Conclusions Given current bioinformatics approaches, there is no single 'best' reference transcriptome for a particular set of raw data. As the optimum transcriptome is a moving target, improving (or not) with new tools and approaches, automated and programmable pipelines are invaluable for managing the computationally-intensive tasks required for re-processing large sets of samples with revised pipelines and ensuring a common evaluation work ow is applied to all samples. Thus, re-assembling existing data with new tools using automated and programmable pipelines may yield more accurate identi cation of taxon-speci c trends across samples in addition to novel and useful products for the community.• Re-assembly with new tools can yield new results • Automated and programmable pipelines can be used to process arbitrarily many samples. • Analyzing many samples using a common pipeline identi es taxon-speci c trends.

Transcriptome of Thalassicolla nucleata Holobiont Reveals Details of a Radiolarian Symbiotic Relationship

“…Dinoflagellate transcripts were then aligned to Brandtodinium nutricula RCC 3387 (MMETSP1462) transcriptome assembly (Johnson et al, 2018) with BLASTN using a 90% identity cutoff and 50% coverage cutoff for the shorter sequence. Transcripts aligned to the B. nutricula transcriptome were considered Brandtodinium sequences.…”

“…Original reads of the B. nutricula RCC3387 (MMETSP1462) transcriptome were downloaded from iMicrobe 1 . Its transcriptome assembly were downloaded from Johnson et al (2018). The same pipeline of read alignment, read count generation, rRNA/chloroplast/mitochondria sequence identification, protein prediction and annotation described above was also applied to the MMETSP1462 transcriptome.…”

Trial by phylogenetics - Evaluating the Multi-Species Coalescent for phylogenetic inference on taxa with high levels of paralogy (Gonyaulacales, Dinophyceae)

“…The stringency for quality trimming of RNA-seq libraries prior to assembly plays a role in determining the number of unique contigs recovered and the subsequent assembly quality of transcriptomes. Regarding the transcriptome assembly method, Johnson et al (2018) evaluated the publicly available assemblies from MMETSP using BUSCO scores, compared to processing and re-assembly with Trinity (Johnson et al, 2018). Johnson et al (2018) demonstrated that while the raw data available from the MMETSP project is an excellent resource, the assemblies available as part of the project are of a lower quality than what can be achieved with current methods (Johnson et al, 2018).…”

“…High stringency is usually favored, however MacManes (2014) found that this can be detrimental to the assembly and the quality cut off scores used in the present study were based on those recommendations (MacManes, 2014). In short, the trimming and assembly pipeline used for the assemblies available as part of MMETSP is no longer state-of-the-art and this is reflected in the quality comparison conducted by Johnson et al (2018). To address this problem, we developed a workflow implementation of current best-practice transcriptome assembly methods as part of this study.…”

Phytochrome evolution in 3D: deletion, duplication, and diversification

“…Arabidopsis and related mustards have five phytochromes, but one of these (PHYD) is a late duplication found only in the flowering plant family Brassicaceae (Mathews, ; Mathews & McBreen, ). The model kelp Ectocarpus siliculosus has four phytochromes (Cock et al , ), as do some Hemiselmis strains (Keeling et al , ; Johnson et al , ). Remarkably, phytochrome gene families have apparently undergone much greater expansion in glaucophytes, with as many as 11 members of this family in Cyanophora paradoxa (Price et al , ).…”