Metatranscriptomics as a tool to identify fungal species and subspecies in mixed communities

Abstract

21High-throughput sequencing (HTS) enables the generation of large amounts of genome 22 sequence data at a reasonable cost. Organisms in mixed microbial communities can now 23 be sequenced and identified in a culture-independent way, usually using amplicon 24 sequencing of a DNA barcode. Bulk RNA-seq (metatranscriptomics) has several 25 advantages over DNA-based amplicon sequencing: it is less susceptible to amplification 26 biases, it captures only living organisms, and it enables a larger set of genes to be used for 27 taxonomic identification. Using a defined mock community comprised of 17 fungal isolates, 28 we evaluated whether metatranscriptomics can accurately identify fungal species and 29 subspecies in mixed communities. Overall, 72.9% of the RNA transcripts were classified, 30 from which the vast majority (99.5%) were correctly identified at the species-level. Of the 15 31 species sequenced, 13 were retrieved and identified correctly. We also detected strain-level 32 variation within the Cryptococcus species complexes: 99.3% of transcripts assigned to 33 Cryptococcus were classified as one of the four strains used in the mock community. 34 Laboratory contaminants and/or misclassifications were diverse but represented only 35 0.44% of the transcripts. Hence, these results show that it is possible to obtain accurate 36 species-and strain-level fungal identification from metatranscriptome data as long as taxa 37 identified at low abundance are discarded to avoid false-positives derived from 38 contamination or misclassifications. This study therefore establishes a base-line for the 39 application of metatranscriptomics in clinical mycology and ecological studies. 40

CCMetagen: comprehensive and accurate identification of eukaryotes and prokaryotes in metagenomic data

“…The KMA indexing of the nt database was limited to only include k-mers with a two-letter prefix, which 179 on average corresponds to only saving non-overlapping k-mers. This prefixing substantially increases 180 the speed and could also be applied to the RefSeq database if faster processing time is required 181 We validated the CCMetagen pipeline with a fungal community previously generated in vitro by 211 culturing, processing and sequencing 15 fungal species (Marcelino et al 2019a, Supplemental Table 212 S2). The analyses were performed using the NCBI nt collection as reference.…”

Metatranscriptomics reveals a shift in microbial community composition and function during summer months in a coastal marine environment

“…While metagenomics profiles the taxonomy of a sample, metatranscriptomics can profile the biological functions that are present in the sample and are active or differentially accumulated in particular environments (Aguiar-Pulido et al 2016). Metatranscriptomics can also be used to deduce taxonomic information for dominant taxa in communities (Shi et al 2011; Neves et al 2017; Marcelino et al 2019), in particular with longer transcriptome contigs, which are expected to produce correct taxonomic assignments (Shakya et al 2019). Metatranscriptomics is typically conducted through next-generation sequencing, which has the benefit of identifying novel genes and functions not known to be used by the identified taxa (Gilbert et al 2008).…”