Tag jumps illuminated--reducing sequence-to-sample misidentifications in metabarcoding studies.
Metabarcoding of environmental samples on second-generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed-template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt-ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina-based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping-derived sequences to samples.
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen K, Denmark.|Center for Zoo and Wild Animal Health, Copenhagen Zoo, 2000, Frederiksberg, Denmark.
Ida Baerholm
IB
Schnell
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen K, Denmark.|School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, UK.
Kristine
K
Bohmann
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, 1350, Copenhagen K, Denmark.|Trace and Environmental DNA Laboratory, Department of Environment and Agriculture, Curtin University, Perth, Western Australia, 6102, Australia.
M Thomas P
MT
Gilbert
D000818:Animals; D002685:Chiroptera / Q000382:microbiology; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D003951:Diagnostic Errors; D004783:Environmental Microbiology; D005243:Feces / Q000382:microbiology; D000069196:Gastrointestinal Microbiome; D007865:Leeches / Q000382:microbiology; D056186:Metagenomics / Q000379:methods*
chimeras; diversity assessment; environmental DNA; metabarcoding; second-generation sequencing; tag jumping