Gender-based variation in the diet of the Pond bat using metabarcoding of dung pellets

Abstract

DNA extraction from faecal pellets We analysed a total of 253 faecal pellets with DNA metabarcoding. A total of 93 pellets did not yield results (31 males and 62 females) and were excluded from further analysis. The remaining pellets were 95 females and 65 males. Details of all pellets analysed are provided in Supplementary Data SD2b. The pellets were ground to a fine powder in liquid nitrogen with a mortar and pestle using the protocol of the commercial Qiagen QIAamp DNA Stool Mini Kit in a special Ancient DNA facility dedicated to work with samples with degraded DNA and following established protocols to avoid contamination such as the inclusion of extraction blanks (Cooper and Poinar 2000). Subsequently, aliquots of each extraction were further purified using Promega PCR purification columns.

PCR amplification of COI and 16S mini-barcodes and library preparation Amplifications of the ~313 bp long mitochondrial COI mini-barcoding marker were performed using forward primer ZBJ-ArtF1c 5’-AGATATTGGAACWTTATATTTTATTTTTGG-3’ and reverse primer ZBJ-ArtR2c 5’- WACTAATCAATTWCCAAATCCTCC-3’ (Zeale et al. 2011). The ~157 bp long mitochondrial 16S barcoding marker was amplified using the forward primer P7_FO-16S 5’- RGACGAGAAGACCCTATARA-3’ and P7_R0-16S 5’-ACGCTGTTATCCCTAARGTA-3’ (Elbrecht et al. 2016). Primers were labelled for DNA metabarcoding with IonExpress labels. The PCR was carried out in 30 microliter reactions containing 0.20 µl Qiagen taq 5u/µl, 3 µl 10x Qiagen buffer, 2 µl 2,5mM dNTP’s, 0,5 µl 10 µM forward primer, 0,5 µl 10 µM reverse primer, 1,5 µl 25mM MgCl2, 0,5 µl 10 mg/ml BSA, 19.80 µl MiliQ and 2 µl template. Amplifications were performed using the following PCR programme: 5 min denaturation at 95°C followed by 40 cycles of 20 seconds denaturation at 95°C, 20 seconds annealing at 50°C and 1-minute elongation at 72°C. Final elongation was conducted at 72°C for 7 minutes on a C1000 Biorad PCR machine.

Ion Torrent sequencing Primer dimer and other contaminants were removed by using 0.9x Ampure XP beads (Agencourt) to which the PCR products were bound. The beads were washed with 150 microliter 70% EtOH twice and resuspended in 20 microliter TE buffer. Cleaned PCR products were quantified using an Agilent 2100 Bioanalyzer DNA High sensitivity chip. An equimolar pool was prepared of the amplicon libraries at the highest possible concentration. This equimolar pool was diluted according to the calculated template dilution factor to target 10-30% of all positive Ion Sphere Particles. Template preparation and enrichment was carried out with the Ion One Touch 200 Template kit with use of the Ion One Touch System, according to the manufacturer's protocol. The quality control of the Ion One Touch 200 Ion Sphere Particles was done with the Ion Sphere Quality Control kit using a Life Qubit 2.0. The enriched Ion Spheres were prepared for sequencing on a Personal Genome Machine (PGM) with the Ion PGM 200 Sequencing kit as described in the protocol and deposited on an Ion-314 chip (520 cycles per run) in three consecutive sequencing runs.

Sequence analysis and data filtering Reads obtained from Ion Torrent sequencing were automatically sorted into separate sequence files based on the MID labels by the Ion Torrent software. The reads were further processed with PRINSEQ (version 0.20.3, Schmieder and Edwards 2011) with the following settings: a minimum read length of 100 bp, trimming to 140 bp, minimum mean quality of Q24 per read, additional trimming of '3 end bases with a Q lower than 24 and removal of full duplicate sequences. Filtered reads were clustered into Operational Taxonomic Units (OTUs) defined by a sequence similarity of at least 97% using CD-HIT-EST (Li and Godzik 2006). Singletons were omitted. For each cluster the representative sequences were BLASTed with the NCBI-blast+ software package (version 2.2.28+, Camacho et al. 2009) against either the NCBI GenBank nucleotide database or a custom database containing all Arthropod sequences located on the Barcode of Life Database (Ratnasingham and Hebert 2007). BLAST hits were filtered according to the following criteria: minimum hit length of a 100 bp, minimum hit similarity of 97% and a maximum e-value of 0.05. Reference databases of Dutch species such as http://www.nederlandsesoorten.nl/ were used to check if a species had been recorded for the Netherlands. All species not (yet) known for The Netherlands were reduced to genus level or to the family level if the genus is also unknown to occur.