Winter diet of Gentoo penguins in South Georgia

See spreadsheets - Gentoo Experiment Details 18sEach number corresponds to each worksheet1. 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 LayoutSamples 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 PCRThe second round PCR is to add sequencing adapters and additional 8 bp MIDs. See sheet for PCR conditions.5. MiseqMiSeq 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 KrillThe 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 /DegenerateThe 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 PCRThe second round PCR is to add sequencing adapters and additional 10 bp MIDs. See sheet for PCR conditions.5. MiseqMiSeq 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.

详见电子表格：巴布亚企鹅实验细节（18S组），编号对应各工作表。

1. 样本与采样时间
巴布亚企鹅粪样采集于南乔治亚州坎伯兰湾的梅维肯（Maiviken）繁殖种群。2018年4月3日至9月19日期间每周开展一次采样，因雪崩风险缺席了6月的一次采样。24次采样中每次采集25份新鲜粪样，总计获得600份样本。使用无菌刮铲将粪样装入盛有80%乙醇的2mL螺旋盖塑料离心管中，并置于-20℃冻存。
采用Promega Maxwell RSC组织DNA提取试剂盒，从约30mg粪便样本中提取基因组DNA。每份提取体系包含软质组分或约500mL乙醇悬浮液。将样本离心沉淀后倾去乙醇，用120μL S.T.A.R缓冲液重悬样本并均质化。取100μL上清液加入第1号孔，最终用100μL TE缓冲液洗脱样本。

2. 板排布
样本按1:10比例稀释后接种至96孔板，每块板均设置阳性对照（鱼、鱿鱼、虾混合DNA）与PCR阴性对照。

3. 第一轮PCR
所有样本采用高度保守的后生动物引物组进行扩增，该引物组可扩增核基因组18S基因的一段区域（McInnes等，2017a）。第一轮PCR用于扩增目标标记序列，并为正向与反向引物添加样本特异性7bp多重识别标签（MID标签）以及Illumina测序引物。PCR反应条件详见对应工作表。

4. 第二轮PCR
第二轮PCR用于添加测序接头以及额外的8bp MID标签。PCR反应条件详见对应工作表。

5. MiSeq测序
使用Illumina MiSeq基因组测序仪，搭配MiSeq V2测序试剂盒（300个循环）进行测序。样本排布、i5与i7接头以及第一轮PCR的MID标签信息详见对应工作表。

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详见电子表格：巴布亚企鹅实验细节（鱼类与磷虾组）

经18S测序分析鉴定出含有猎物DNA序列的粪样共222份，本部分采用另外两对引物对其进行检测，以实现鱼类与磷虾的物种水平鉴定。

1. 样本
猎物DNA呈阳性的样本及其板排布信息

2. 与3. 第一轮PCR（磷虾/简并引物体系）
第一轮PCR用于扩增目标标记序列，并为正向与反向引物添加样本特异性6bp多重识别标签（MID标签）以及Illumina测序引物。PCR反应条件详见对应工作表。

4. 第二轮PCR
第二轮PCR用于添加测序接头以及额外的10bp MID标签。PCR反应条件详见对应工作表。

5. MiSeq测序
使用Illumina MiSeq基因组测序仪，搭配MiSeq V2测序试剂盒（300个循环）进行测序。样本排布、正向与反向接头以及第一轮PCR的MID标签信息详见对应工作表。

本研究作为AAS 4556号项目的一部分完成。