*5.4. Multiplexing and Library Preparation*

The ability to scale up metabarcoding studies relies on the use of sample-specific labels in the form of unique sequences of nucleotides which are attached to amplicons. These

unique identifiers allow hundreds or thousands of samples to be pooled for sequencing (multiplexing), significantly increasing the capacity of one sequencing run. Methods for indexing of samples occur either during the initial PCR amplification through nucleotide additions to amplicons or through a secondary PCR amplification along with adapters to allow successful sequencing (library indices) (reviewed in [146]). Each of the methods comes with trade-offs between many factors, mainly the risk of cross-contamination, efficiency of PCR amplification, and overall cost [146]. The two-step PCR approach is most widely used in pollen metabarcoding studies (Table S1), allowing a cost-effective approach to sample labelling whilst allowing effective detection of cross-contamination, but comes with the caveat of increased risk of biases due to an additional amplification stage [146].
