Increased throughput for meta-barcoding of mixed pollen samples using a dual-index approach with the Illumina MiSeq platform

1. Next generation sequencing of mixed pollen samples constitutes a suitable alternative for conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods. 2. We thus enhanced these by a dual-indexing strategy for plant meta-barcoding with the marker ITS2, which is highly multiplexable and does not require further adapter ligation steps after amplification. 3. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, samples were sufficiently covered with reads to assess the complete diversity of the samples. 4. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number plant taxa by 30% to 76,336. 5. This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.