We use RNA sequencing to investigate which genetic/physiological pathways in Antarctic krill are affected by increased CO2 levels. We carried out larval CO2 exposure experiments in March 2012 at the AAD aquarium. Two developmental stages were used (Calyptopis I and Furcilia V) and three CO2 levels (control, 1000 and 2000 ppm). These were short term experiments (2 days) - since initial longer experiments starting with fertilized eggs resulted in differences in developmental stages between treatments and control which could confound the data. RNA was extracted from larvae and high-throughput RNA sequencing (RNA-seq) was carried out on 6 samples (2 stages * 3 treatments). Sequencing was carried out on an Illumina sequencer (Genome Analyzer II). We collected ~ 60 million sequence reads per sample (Data in FASTA format each read gives 100 base pairs of sequence), so a total of ~360 million reads (36 billion bp of data).

1st Experiment 24/11/16 ************************************************************************************************ See 2016_11_24_Miseq_Sheet 1. Sanger Sequencing Plate #4 - 25mg of Tissue was extracted by AGRF. DNA was diluted to 5ng/ul. Samples were sanger sequenced with 16SAR (Palumbi) primer. If they failed, I used COI3 cocktail (Ivanova). FASTA sequences from Plate 4 are in the folder named Sanger Sequence FASTA Plate #4. Naming - Plate position, primer, sample ID. ie reater than A1-16S-AR_1952. 2. DNA and Tissue Pools of Plate 4 We wanted to explore the possibility of using a metabarcoding approach. For metabarcoding we re-examined specimens already identified from sanger sequences. We mixed DNA from many samples (n=16 or n=96) and did a single amplification (i.e. up to 96 DNA extractions processed in a single-tube marker amplification). We also took it a step further and tried blending a set amount of tissue from many fish specimens (n=16 or n=96) and did a single DNA extraction on the tissue mixes (i.e. a single DNA extraction and single tube amplification for up to 96 samples). See 2016_11_24_Miseq_Sheet for DNA and Tissue Pool mixes. 3. Miseq Run 16 samples were ran on a 250bp pe read. Each sample was amplified with 3 primer sets - COI (please note one dual labelled set was used), 12s and 16s (Primers listed on 2016_11_24_Miseq_Sheet). They were diluted 1:10 and illumina sequencing adaptors were added (please note I used same I7 and I5 per sample, so they had to be sorted on amplicon). 2016_11_24_fastq_files has the data from miseq. and 2016_11_24_merged_fastq_files has the merged files. For some unknown reason 16s tissue produced no data. 2nd Experiment 04/07/17 ************************************************************************************************* 1. DNA Extractions Plate #1, 2 and 3 - 25mg of Tisse was extracted by AGRF. DNA was diluted to 5ng/ul. We also used Plate #4 from experiment above. See Plate Layout for sample allocation. 2. Tissue and DNA Pools DNA pools were from Plate 1, 2, 3 and 4. Tissue Mixes were from Plate 2 and 4 only. We wanted to explore the possibility of using a metabarcoding approach. We mixed DNA from many samples (n=16 or n=96) and did a single amplification (i.e. up to 96 DNA extractions processed in a single-tube marker amplification). We also took it a step further and tried blending a set amount of tissue from many fish specimens (n=16 or n=96) and did a single DNA extraction on the tissue mixes (i.e. a single DNA extraction and single tube amplification for up to 96 samples). See plate layout for DNA and Tissue Pool mixes. 3. Miseq Run 577 samples were sequenced in a 250bp pe read. See 2017_07_04_Miseq Sheet. Plate 1, 2 3 and 4 were all sequenced with Leray Primers.(Please note I accidentally amplified the first half of plate one with one pair of dual labelled COI primers, index on miseq sheet). I also made a plate of tissue and DNA pools (see plate layout for DNA and Tissue Pool mixes) and amplified those with 4 primers (primer sequences on miseq sheet) COI (individual dual labelled primers, 1st round index are on miseq sheet) 12s Fish 16s Chordate NADH The last 4 samples with 12s were to add to database as there are no 12S sequences for those species on genbank. See PCR recipes for annealing temp and cycling etc I accidentally put the marker under sample name so the original sample ID was lost and miseq gave it a new name (name from miseq output) and then another new name from merged file. Finally I gave them a unique sample ID. See name file if you need more information. 2017_07_04 has the data from miseq. and 2017_07_04_merged_fastq_files has the merged files. Samples were clustered using zero radius OTU's. 4.Results See Results database. The spreadsheet has all of the possible name combinations from the run. It also contains the Haul ID and date, time, lat, long etc. There is a morph taxa ID which refers to what the observer has identified the fish and then there is Seq_Taxa_ID which is the sequencing result. There is also a list of primers that were used to identify the fish. 0 indicated that the primer wasnt used, 1 indicates it was. The second tab has all of the info for the samples that failed. *************************************************************************************************

From the abstract of the referenced paper: Randomly amplified polymorphic DNA markers (RAPDs) were applied in a cephalopod population study. Samples of the squid Moroteuthis ingens taken from around the Falkland Islands and Macquarie Island were used to test a null hypothesis that M. ingens forms a single, panmictic population in the Southern Ocean. Six of the 8 arbitrary RAPD primers screened produced a total of 30 reproducible polymorphic bands. Analysis of RAPD allele frequencies demonstrated high levels of variation between individuals but little variation between two sample sites. Although the differentiation between the two sites was low, subtle population structure was detected and the null hypothesis was rejected. The implications of low genetic differentiation between the two sites are briefly discussed in terms of possible egg and para-larval drift facilitated via the circumpolar current. The PDF document available for download also includes a number of data tables. See also ASAC project 1242 (ASAC_1242), Biology of Southern Ocean squid, ecological importance and potential commercial implications - a preliminary assessment.

Metadata record for data collected as part of Australian Antarctic Science project 3010 in the Australian Antarctic program. From the abstract of the referenced paper: The evolutionary history of Antarctic organisms is becoming increasingly important to understand and manage population trajectories under rapid environmental change. The Antarctic sea spider Nymphon australe, with an apparently large population size compared with other sea spider species, is an ideal target to look for molecular signatures of past climatic events. We analysed mitochondrial DNA of specimens collected from the Antarctic continent and two Antarctic islands (AI) to infer past population processes and understand current genetic structure. Demographic history analyses suggest populations survived in refugia during the Last Glacial Maximum. The high genetic diversity found in the Antarctic Peninsula and East Antarctic (EA) seems related to multiple demographic contraction-expansion events associated with deep-sea refugia, while the low genetic diversity in the Weddell Sea points to a more recent expansion from a shelf refugium. We suggest the genetic structure of N. australe from AI reflects recent colonization from the continent. At a local level, EA populations reveal generally low genetic differentiation, geographically and bathymetrically, suggesting limited restrictions to dispersal. Results highlight regional differences in demographic histories and how these relate to the variation in intensity of glaciation-deglaciation events around Antarctica, critical for the study of local evolutionary processes. These are valuable data for understanding the remarkable success of Antarctic pycnogonids, and how environmental changes have shaped the evolution and diversification of Southern Ocean benthic biodiversity.