Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

Abstract

DNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most wellestablished bioinformatics tools for DNA metabarcoding were originally designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the 5 10 2 eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the crossvalidation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.

Fungi in aquatic ecosystems

“…Although not yet isolated into culture, these organisms may be highly abundant and dominate natural ecosystems 29 . In TABLE S4 of REF 30 it is obvious that most fungi in a large number of lakes and ponds in northeast Germany are uncharacterized and hence unknown. A similar pattern has been also found by another study exploring biogeographical patterns and biases of fungal diversity 31 .…”

“…This 'annotation gap' does not only affect functional genes, but is even clearly visible in curated fungal barcode reference data. Currently, attempts have been made towards unifying all ribosomal regions into one single ribosomal fungal marker that covers all of the eukaryotic ribosomal regions at once 30,47 , which greatly improves taxonomic resolution of non-cultured and unknown fungi. However, we want to highlight that there is a great need for increased cultivation of fungal strains both for phylogenetic and physiological characterization which allows for linking phylogenetic, morphological as well as physiological features in a reliable manner.…”

Can we use environmental DNA as holotypes?

“…Recently, the cost of analyzing the DNA samples with NGS has also dropped significantly, thus boosting the potential for using this technique (van Dijk et al 2014). Long-read sequencing has become currently available at high quality by using nanopore or PacBio sequencing (Heeger et al 2018;Wurzbacher et al 2018). By detecting hundreds to thousands of fungal OTUs in hundreds of samples within weeks, metabarcoding approaches tear down the limits of cultivation-based approaches in community ecology studies (Peršoh 2015).…”

“…DNA metabarcoding has therefore become an important tool and is now commonly used to understand species diversity and community structure in complex communities of microorganisms (Heeger et al 2018;Jayawardena et al 2018b). The approach is based on the DNA-barcoding concept and uses the massive amount of sequence data produced through high-throughput sequencing (HTS) techniques.…”

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale

“…A solution to sequence the resulting long amplicons is offered by recent developments in third generation sequencing platforms, which now enable researchers to generate ultra-long reads, of up to 800 kb [21]. Recently, amplicons of several kilobases of the rDNA cluster were sequenced using Pacific Bioscience (PacBio) technology, to explore fungal community composition [22,23]. But while PacBio sequencing is well suited for long amplicon sequencing, it is currently not readily available to every laboratory due to the high cost and limited distribution of sequencing machines.…”