Capture probe, metabarcoding, or shotgun sequencing: which best reflects local vegetation?

Metabarcoding is the most widely applied method for studying plant communities using environmental DNA, with shotgun sequencing and capture probes being alternative methods. Any method's ability to detect and correctly identify plant taxa may vary with DNA preservation, DNA reference library and the size of the local flora, making it difficult to compare results from different environments. Here we compare these methods using lake surface-sediments from Northern Fennoscandia with the PhyloNorway genome skim reference library (1500 taxa) that includes almost all species of the regional flora. We also undertook vegetation surveys from around the lakes to estimate the true positive detection rate, identify false positive detections, and provide optimal filtering cut-off thresholds for the three methods. Optimal filter cutoffs were one PCR replicate and 3 reads for metabarcoding, and 3.2x10e-6 and 5.6x10e-6 of queried reads, respectively, for the shotgun and capture probe data. Applying these thresholds, the rate of false positives was too high for reliable identification at species level based on shotgun (49%) and capture probes (62%), whereas it was low for metabarcoding (5-12%). All methods were reliable at genus and family levels after applying the optimal filtering thresholds (<4% false positives). Our results show that metabarcoding on average detects 2.1 times as many true positive taxa as shotgun sequencing, and 6.4 times as many taxa as capture probes. Proportions of filtered metabarcoding and shotgun reads were significantly related to abundance categories from vegetation surveys, but this was not the case for capture probe data. We expect the false positive rate of shotgun sequencing to decrease with increasing genome completeness in the reference libraries, and be advantageous for highly degraded DNA with fragments too short for metabarcoding. At present, metabarcoding provides the highest detectability and taxonomic resolution for correct identification of vascular plants.