Towards PacBio‐based pan‐eukaryote metabarcoding using full‐length ITS sequences

Tedersoo, Leho; Anslan, Sten

doi:10.1111/1758-2229.12776

Cited by 80 publications

(62 citation statements)

References 49 publications

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“…The purified DNA extracts were subjected to amplification with the universal eukaryotic primers ITS9mun and ITS4ngsUni that have been nominated for global microbiome analyses of eukaryotes at species-level resolution (Tedersoo and Lindahl, 2016;Tedersoo and Anslan, 2019). These primers amplify 170 bp of the 18S rRNA gene and full-length Internal Transcribed Spacer (ITS), which serves as an official barcode for fungi (Schoch et al, 2012;Nilsson et al, 2018) and performs well on many groups of soil animals and protists (Coleman, 2009;Pawlowski et al, 2012).…”

Section: Molecular Analysis and Bioinformaticsmentioning

confidence: 99%

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe

et al. 2020

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Section: Molecular Analysis and Bioinformaticsmentioning

confidence: 99%

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe

et al. 2020

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“…The possibility of long-read metabarcoding was also recently explored (Callahan et al 2019;Krehenwinkel et al 2019a;Tedersoo and Anslan 2019). Barcode sequences of several thousand base pairs for a whole community would greatly improve the phylogenetic resolution of metabarcoding and allow community-level phylogenetic analysis.…”

Section: Environmental Dna Metabarcoding Of Spidersmentioning

confidence: 99%

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

et al. 2020

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Large-scale studies on community ecology are highly desirable but often difficult to accomplish due to the considerable investment of time, labor and, money required to characterize richness, abundance, relatedness, and interactions. Nonetheless, such large-scale perspectives are necessary for understanding the composition, dynamics, and resilience of biological communities. Small invertebrates play a central role in ecosystems, occupying critical positions in the food web and performing a broad variety of ecological functions. However, it has been particularly difficult to adequately characterize communities of these animals because of their exceptionally high diversity and abundance. Spiders in particular fulfill key roles as both predator and prey in terrestrial food webs and are hence an important focus of ecological studies. In recent years, large-scale community analyses have benefitted tremendously from advances in DNA barcoding technology. High-throughput sequencing (HTS), particularly DNA metabarcoding, enables community-wide analyses of diversity and interactions at unprecedented scales and at a fraction of the cost that was previously possible. Here, we review the current state of the application of these technologies to the analysis of spider communities. We discuss amplicon-based DNA barcoding and metabarcoding for the analysis of community diversity and molecular gut content analysis for assessing predator-prey relationships. We also highlight applications of the third generation sequencing technology for long read and portable DNA barcoding. We then address the development of theoretical frameworks for community-level studies, and finally highlight critical gaps and future directions for DNA analysis of spider communities.

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“…Recently, Tedersoo et al . (2018) and Tedersoo & Anslan (2019) used Pacific Biosciences (PacBio) to sequence longer fragments (1200–2500 bases), which can provide better taxonomic resolution (Singer et al ., 2016), but the PacBio platform has also been used to sequence shorter ITS (800–900 bases: Redondo et al ., 2018) and ITS2 markers (200–450 bases: Kyaschenko et al ., 2017a,b; Varenius et al ., 2017; Castaño et al ., 2018; Sterkenburg et al ., 2018). Despite higher error rates of single reads for PacBio ( c. 11%) as compared with Illumina MiSeq ( c. 0.1–2.6%) (May et al ., 2015; Pfeiffer et al ., 2018), the PacBio SMRT technology enables correction of random errors by calculating a consensus sequence based on typically > 30 separate sequencing passes, reducing errors to < 1% when short fragments are sequenced (Reuter et al ., 2015).…”

Section: Introductionmentioning

confidence: 99%

Optimized metabarcoding with Pacific biosciences enables semi‐quantitative analysis of fungal communities

et al. 2020

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Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

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Towards PacBio‐based pan‐eukaryote metabarcoding using full‐length ITS sequences

Cited by 80 publications

References 49 publications

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

Optimized metabarcoding with Pacific biosciences enables semi‐quantitative analysis of fungal communities

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