June 2018 Adélie penguin scats were collected from Signy Island (South Orkney Islands) during crèche (December/January) 2014/15 and 2015/16 and stored in 80% Ethanol. DNA was extracted from ~30 mg of faecal material using a Promega ‘Maxwell 16' instrument and a Maxwell® 16 Tissue DNA kit. A total of 450 samples were analysed: 30 extractions per week for 2015 and 60 per week for samples collected in 2016. Three DNA markers providing different taxonomic information were amplified from penguin faecal DNA. First, ALL faecal DNA samples were characterised using a highly conserved metazoan primer set that amplifies a region of the nuclear 18S gene. In addition, a subset of faecal samples from each year were also characterised with two other primer pairs that amplify a region of the mtDNA 16S gene to allow species-level identification for most fish (16S_Fish) and krill (16S_Krill) species respectively. During amplification of markers, the products were tagged with a unique pair of index primers allowing samples to be pooled and sequenced (2x150bp) on a MiSeq high-throughput DNA sequencer. - See Adelie Pengiun Diet CCAMLR paper for all of the primer/PCR details - See BAS Adelie 18s Krill and Fish subset excel spreadsheet for sample details. - See BAS Adelie 18s ALL samples fastq for 18s fastq files - See BAS Adelie 16s Krill subset fastq for 16s krill fastq files - See BAS Adelie Fish subset fastq for 16s fish fastq files ##################################################################################### November 2018 In addition we also amplified all 450 samples with the 16S_Fish marker. - See Adelie Experiment Details 16s Fish for sample details, plate layout, first and second round PCR and miseq sheet. - See BAS Adelie Fish ALL Samples fastq for 16s fish fastq files

See spreadsheets - Gentoo Experiment Details 18s Each number corresponds to each worksheet 1. Samples and Date Gentoo penguin scats were collected from Cumberland Bay, South Georgia (from the Maiviken colony). Visits were made weekly between 3 April and 19 Sep 2018 (one visit in June was missed owing to avalanche risk). During each of the 24 visits, 25 fresh scats were collected, producing a total of 600 samples. Samples were scooped into a 2 ml plastic screw-top tube containing 80% ethanol with a clean spatula and frozen at -20 degrees. DNA was extracted from ~30 mg of faecal material using the Promega Maxwell RSC Tissue DNA Kit. Each extraction contained a soft part or ~500ml of EtOH slurry. The samples were spun down, the EtOH was poured off, the sample was re-suspended in 120ul of S.T.A.R buffer and homogenised. 100ul of the supernatant was added to well number 1 and samples were eluted in 100ul of TE. 2. Plate Layout Samples were diluted 1/10 and plated out on 96 well plates. Each plate had a positive control (fish, squid, shrimp DNA mix) and a negative PCR control. 3. 1st Round PCR. All samples were analysed using a highly conserved metazoan primer set that amplifies a region of the nuclear 18S gene ( McInnes et al. 2017a). The first round PCR is to amplify the target marker and add sample-specific (7bp) multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers. See sheet for PCR conditions. 4. 2nd Round PCR The second round PCR is to add sequencing adapters and additional 8 bp MIDs. See sheet for PCR conditions. 5. Miseq MiSeq genome sequencer (Illumina), using the MISEQ V2 reagent kits (300 cycles). See sheet for sample layout, i5 and i7 adapters and first round MID tags ########################################################################################### See spreadsheet - Gentoo Experiment Details Fish and Krill The scat samples containing prey DNA sequences from the 18S analysis (n=222) were characterised with two other primer pairs allow species-level identification for fish and krill. 1. Samples The samples positive for prey DNA and their plate layout 2. and 3. 1st Round PCR Krill /Degenerate The first round PCR is to amplify the target marker and add sample-specific (6bp) multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers. See sheet for PCR conditions. 4. 2nd Round PCR The second round PCR is to add sequencing adapters and additional 10 bp MIDs. See sheet for PCR conditions. 5. Miseq MiSeq genome sequencer (Illumina), using the MISEQ V2 reagent kits (300 cycles). See sheet for sample layout, R and F adapters and first round MID tags. This work was completed as part of AAS project 4556.