Our understanding of trophic interactions of small insectivorous mammals has been drastically improved with the advent of DNA metabarcoding. The technique has continued to be optimised over the years, with primer choice repeatedly being a vital factor for dietary inferences. However, the majority of dietary studies examining the effect of primer choice often rely on in silico analyses or comparing between species that occupy an identical niche type. Here we apply DNA metabarcoding to empirically compare the prey detection capabilities of two widely used primer sets when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus hipposideros) and two ground dwelling insectivores (greater white-toothed shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R. hipposideros primarily rely on two prey orders (Lepidoptera and Diptera), the unique taxa detected by each primer shows that a combination of primers may be the best approach to fully describe bat trophic ecology. However, random forest classifier analysis suggest that one highly degenerate primer set detected the majority of both shrews’ diet despite higher levels of host amplification. The wide range of prey consumed by ground-dwelling insectivores can therefore be accurately documented from using a single broad-range primer set, which can decrease cost and labour. The results presented here show that dietary inferences will differ depending on the primer or primer combination used for insectivores occupying different niches (i.e. hunting in the air or ground) and demonstrate the importance of performing empirical pilot studies for novel study systems.

This is a metabarcoding dataset for Browett SS, Curran TG, O’Meara DB, Harrington AP, Sales NG, Antwis RE, O’Neill D, McDevitt AD (2021) Primer biases in the molecular assessment of diet in multiple insectivorous mammals. BioRxiv, Doi: 10.1101/2021.01.18.426998

DNA was extracted from faeces of 24 bats (Rhinolophus hipposideros) and gut contents of 27 shrews (Sorex minutus and Crocidura russula). The COI region was amplified using 2 primer sets, referred to as Gillet and Zeale. There are four multiplexed fastq files. A forward and reverse for the 24 bats and a forward and reverse for the 27 shrews. Samples were multiplexed using primers with dual-index 8 base pair MID tags on them. The sequences contain the primer region and MID tags. A file with the list of tags and primer sequence is included. Sequencing adapters were ligated using a PCR-free method. Library was sequenced on an Illumina MiSeq V2 300 cycle kit.

For full details on data collection, preparation and sequencing, see https://doi.org/10.1101/2021.01.18.426998 (and forwarding link to published article) and https://github.com/ShrewlockHolmes/

Further information on samples is also available from https://github.com/ShrewlockHolmes/

The list containing the MID tags and individual sample names are included. The columns are

1: Primer used

2: Sample ID

3: Forward MID tag

4: Reverse MID tag

5: Forward primer sequence

6: Reverse primer sequence

7:9 misc info required for pipeline used for article.