Scraping the bottom of the barrel: are rare high throughput sequences artifacts?. (February 2015)

• Record Type: Journal Article
• Authors: Brown, Shawn P.; Veach, Allison M.; Rigdon-Huss, Anne R.; Grond, Kirsten; Lickteig, Spencer K.; Lothamer, Kale; Oliver, Alena K.; Jumpponen, Ari
• Is Part Of: Fungal ecology. Volume 13(2015:Feb.)
• Page Start: 221
• Page End: 225
• Publication Date: 2015-02
• Subjects: Fungi -- High-throughput sequencing -- Rare biosphere -- Singleton; Fungi -- Ecology -- Periodicals; Mycology -- Periodicals; 579.517
• DOI: 10.1016/j.funeco.2014.08.006
• Languages: English
• ISSNs: 1754-5048

Abstract

Abstract: Metabarcoding data generated using next-generation sequencing (NGS) technologies are overwhelmed with rare taxa and skewed in Operational Taxonomic Unit (OTU) frequencies comprised of few dominant taxa. Low frequency OTUs comprise a rare biosphere of singleton and doubleton OTUs, which may include many artifacts. We present an in-depth analysis of global singletons across sixteen NGS libraries representing different ribosomal RNA gene regions, NGS technologies and chemistries. Our data indicate that many singletons (average of 38 % across gene regions) are likely artifacts or potential artifacts, but a large fraction can be assigned to lower taxonomic levels with very high bootstrap support (∼32 % of sequences to genus with ≥90 % bootstrap cutoff). Further, many singletons clustered into rare OTUs from other datasets highlighting their overlap across datasets or the poor performance of clustering algorithms. These data emphasize a need for caution when discarding rare sequence data en masse : such practices may result in throwing the baby out with the bathwater, and underestimating the biodiversity. Yet, the rare sequences are unlikely to greatly affect ecological metrics. As a result, it may be prudent to err on the side of caution and omit rare OTUs prior to downstream analyses.