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AAS_4556_Adelie_Diet_Mawson_DNA

Adélie penguin and snow petrel scats were collected at Béchervaise Island (67°35’S, 62°49’E) in the austral summers 2014/2015, 2017/2018 and 2019/2020 and stored in 80% Ethanol. DNA was extracted using the Maxwell RSC48 instrument with the Maxwell RSC 48 Tissue DNA Kit (Promega). ~30 mg of the scat was added to 250 μL of S.T.A.R buffer (Roche Diagnostics). All remaining steps followed the manufacturer’s instructions. Reagent blank controls (n=5) were added to the extraction process. DNA was plated out and diluted 1:5. In total, there were 465 scat samples; 302 collected from Adélie penguins and 163 from snow petrels. Three DNA markers providing different taxonomic information were amplified. 1. 18s - All samples (n=480 includes positives and negatives) were amplified with a primer set that amplifies ~170bp of the nuclear 18S gene (McInnes et al., (2017b) DNA Metabarcoding as a Marine Conservation and Management Tool: A Circumpolar Examination of Fishery Discards in the Diet of Threatened Albatrosses. Frontiers in Marine Science). A PNA clamp was also added to suppress bird and mammal DNA . 2. Krill - We characterised the taxonomic identity of krill by amplifying ~250bp of the 16S rDNA gene (Ratcliffe et al, (2021), Changes in prey fields increase the potential for spatial overlap between gentoo penguins and a krill fishery within a marine protected area. Diversity and Distributions). Samples were considered positive for krill if the Ct value was less than 35 (n=120). PCR amplifications were performed in two rounds, the first to amplify the target gene and add sample-specific 6 or 7 bp multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers, the second to add sequencing adapters and additional 8 bp MIDs. PCR products from all samples including the blanks, positive and negative controls (n=600) were pooled and purified using Agencourt Ampure (Beckman Coulter, USA) magnetic beads. The pool was diluted to 2 nM and paired-end reads generated on a MiSeq (Illumina, San Diego, CA, USA) with a MiSeq Reagent Kit V2 (1 x 150 bp). The 480 18S_SSU and 120 positive 16S_Krill were sequenced on one chip (n=600). -See 18s and Krill PCR excel sheet for samples, primers, 1st round PCR with MID tags, second round PCR with MID tags and miseq sheet. -See 18s and Krill folder for Fastq files 3. In addition we also amplified 500 samples (465 scats, 17 repeats, extraction blanks, positives and negative controls) with the 16S_Fish marker (Deagle, et al (2007) Studying Seabird Diet through Genetic Analysis of Faeces: A Case Study on Macaroni Penguins (Eudyptes chrysolophus). PLOS ONE 2:e831). PCR amplifications were performed in two rounds, the first to amplify the target gene and add sample-specific 6 bp multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers, the second to add sequencing adapters and additional 8 bp MIDs. PCR products from all samples including the blanks, positive and negative controls (n=500) were pooled and purified using Agencourt Ampure (Beckman Coulter, USA) magnetic beads. The pool was diluted to 2 nM and paired-end reads generated on a MiSeq (Illumina, San Diego, CA, USA) with a MiSeq Reagent Kit V2 (1 x 150 bp). -See Fish PCR excel sheet for samples, primers, 1st round PCR with MID tags, second round PCR with MID tags and miseq sheet. -See Fish Fastq folder for Fastq files The sex of each sample was determined with a real-time melt curve analysis (Faux et al, (2014) High-throughput real-time PCR and melt curve analysis for sexing Southern Ocean seabirds using fecal samples. Theriogenology 81:870-874). Known male and female Adélie penguin, snow petrel samples and Gentoo penguin samples were included on each run. Sexing reaction mix contained 1 μM for each forward and reverse primer, 2 μg BSA, 1 x LightCycler 480 Probes Master (Roche), 1 x EvaGreen (Biotium). Thermal cycling conditions were 95 degrees for 5 min; followed by 40 cycles of 95 degrees for 10s, 55 degrees for 30s and 72 degrees for 10s. Melt curve conditions were 55-95 degrees at a ramp rate of 2.2 C/s with 5 acquisitions per degree. As well as AAS project 4556, these data were also collected as part of AAS 4518.