The Effect of Input DNA Copy Number on Genotype Call and Characterising SNP Markers in the Humpback Whale Genome Using a Nanofluidic Array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue

Local drivers of change in Southern Ocean ecosystems: Human activities and policy implications

Local drivers are human activities or processes that occur in specific locations, and cause physical or ecological change at the local or regional scale. Here, we consider marine and land-derived pollution, non-indigenous species, tourism and other human visits, exploitation of marine resources, recovery of marine mammals, and coastal change as a result of ice loss, in terms of their historic and current extent, and their interactions with the Southern Ocean environment. We summarise projected increases or decreases in the influence of local drivers, and projected changes to their geographic range, concluding that the influence of non-indigenous species, fishing, and the recovery of marine mammals are predicted to increase in the future across the Southern Ocean. Local drivers can be managed regionally, and we identify existing governance frameworks as part of the Antarctic Treaty System and other instruments which may be employed to mitigate or limit their impacts on Southern Ocean ecosystems

The retrospective analysis of Antarctic tracking data project

The Retrospective Analysis of Antarctic Tracking Data (RAATD) is a Scientific Committee for Antarctic Research project led jointly by the Expert Groups on Birds and Marine Mammals and Antarctic Biodiversity Informatics, and endorsed by the Commission for the Conservation of Antarctic Marine Living Resources. RAATD consolidated tracking data for multiple species of Antarctic meso- and top-predators to identify Areas of Ecological Significance. These datasets and accompanying syntheses provide a greater understanding of fundamental ecosystem processes in the Southern Ocean, support modelling of predator distributions under future climate scenarios and create inputs that can be incorporated into decision making processes by management authorities. In this data paper, we present the compiled tracking data from research groups that have worked in the Antarctic since the 1990s. The data are publicly available through biodiversity.aq and the Ocean Biogeographic Information System. The archive includes tracking data from over 70 contributors across 12 national Antarctic programs, and includes data from 17 predator species, 4060 individual animals, and over 2.9 million observed locations

The retrospective analysis of Antarctic tracking data project

The Retrospective Analysis of Antarctic Tracking Data (RAATD) is a Scientific Committee for Antarctic Research project led jointly by the Expert Groups on Birds and Marine Mammals and Antarctic Biodiversity Informatics, and endorsed by the Commission for the Conservation of Antarctic Marine Living Resources. RAATD consolidated tracking data for multiple species of Antarctic meso- and top-predators to identify Areas of Ecological Significance. These datasets and accompanying syntheses provide a greater understanding of fundamental ecosystem processes in the Southern Ocean, support modelling of predator distributions under future climate scenarios and create inputs that can be incorporated into decision making processes by management authorities. In this data paper, we present the compiled tracking data from research groups that have worked in the Antarctic since the 1990s. The data are publicly available through biodiversity.aq and the Ocean Biogeographic Information System. The archive includes tracking data from over 70 contributors across 12 national Antarctic programs, and includes data from 17 predator species, 4060 individual animals, and over 2.9 million observed locations.Supplementary Figure S1: Filtered location data (black) and tag deployment locations (red) for each species. Maps are Lambert Azimuthal projections extending from 90° S to 20° S.Supplementary Table S1: Names and coordinates of the major study sites in the Southern Ocean and on the Antarctic Continent where tracking devices were deployed on the selected species (indicated by their 4-letter codes in the last column).Online Table 1: Description of fields (column names) in the metadata and data files.Supranational committees and organisations including the Scientific Committee on Antarctic Research Life Science Group and BirdLife International. National institutions and foundations, including but not limited to Argentina (Dirección Nacional del Antártico), Australia (Australian Antarctic program; Australian Research Council; Sea World Research and Rescue Foundation Inc., IMOS is a national collaborative research infrastructure, supported by the Australian Government and operated by a consortium of institutions as an unincorporated joint venture, with the University of Tasmania as Lead Agent), Belgium (Belgian Science Policy Office, EU Lifewatch ERIC), Brazil (Brazilian Antarctic Programme; Brazilian National Research Council (CNPq/MCTI) and CAPES), France (Agence Nationale de la Recherche; Centre National d’Etudes Spatiales; Centre National de la Recherche Scientifique; the French Foundation for Research on Biodiversity (FRB; www.fondationbiodiversite.fr) in the context of the CESAB project “RAATD”; Fondation Total; Institut Paul-Emile Victor; Programme Zone Atelier de Recherches sur l’Environnement Antarctique et Subantarctique; Terres Australes et Antarctiques Françaises), Germany (Deutsche Forschungsgemeinschaft, Hanse-Wissenschaftskolleg - Institute for Advanced Study), Italy (Italian National Antarctic Research Program; Ministry for Education University and Research), Japan (Japanese Antarctic Research Expedition; JSPS Kakenhi grant), Monaco (Fondation Prince Albert II de Monaco), New Zealand (Ministry for Primary Industries - BRAG; Pew Charitable Trusts), Norway (Norwegian Antarctic Research Expeditions; Norwegian Research Council), Portugal (Foundation for Science and Technology), South Africa (Department of Environmental Affairs; National Research Foundation; South African National Antarctic Programme), UK (Darwin Plus; Ecosystems Programme at the British Antarctic Survey; Natural Environment Research Council; WWF), and USA (U.S. AMLR Program of NOAA Fisheries; US Office of Polar Programs).http://www.nature.com/sdataam2021Mammal Research Institut

Genotyping analysis workflow with and without STA.

<p><b>[A]</b> Steps 1–5 (denoted in red arrows) correspond to TaqMan^® SNP-GT protocol without STA or following simplex or multiplex STA. <b>[B]</b> Steps 1, 6–9 corresponds to STA reaction setup in simplex and multiplex conditions. Post STA, the amplified products are pooled (simplex STA), or further diluted 5 or 20 fold (multiplex STA) prior to performing TaqMan^® SNP-GT setup using steps 2–5.</p

Effect of DNA copy number on reliability of genotype call data for a heterozygous human genomic DNA sample, NA17316.

<p> <i>Call rate and call accuracy (%) at different number of copies per reaction chamber and for pq calls were determined from 144 data points.</i></p><p> <i>⁽¹⁾% Call rate [All Calls]  = 100*[(Total number of calls) / (Total number of calls + No Calls)].</i></p><p> <i>⁽²⁾% Call rate [pq Calls]  =  100*[(Correct Calls) / (Total number of calls + No Calls)].</i></p><p> <i>⁽³⁾% Call accuracy [pq Calls]  = 100*[(Correct Calls) / (Total number of calls)].</i></p

Summary genotype calls obtained for representative whale DNA samples.

<p>Genotype calls obtained for representative whale DNA samples extracted using CTAB or Maxwell^® tissue extraction kit and following simplex or multiplex STA using either 15 [A] or 45 SNP-GT assays [B]. The genotype call (<b>pp</b>, <b>qq</b> and <b>pq</b>) for each reaction are denoted in ‘<b>red</b>’, ‘<b>green</b>’ and ‘<b>blue</b>’, respectively. ‘+’ refers to samples extracted using CTAB method; '*' refers to each sample extracted using both CTAB and Maxwell^® tissue extraction kit; ‘♦’ refers to concordance with true genotype determined at AAD using an independent method. Note: Regardless of the approach used, genotypes for representative samples using either 15 or 45 SNP-GT assays were the same, as indicated by the same color. Samples EG09-004, EVH09-53, WA07-006 and WA07-003, extracted using CTAB or Maxwell^® tissue extraction kit were genotyped using 15 SNP-GT assays with and without STA and showed a 100% call rate and concordance (data not shown).</p

Samples analysed using the SNP-GT nanofluidic system.

<p>Samples analysed using the SNP-GT nanofluidic system.</p

Effect of reaction DNA copy number on genotype call accuracy for a heterozygous (pq) sample.

<p>Call map view <b>[A]</b> and scatter plots <b>[B]</b> of the genotype calls from reaction chambers containing predicted 38, 18, 7, 4 and 1 copies(y). The genotype call (<b>pp</b>, <b>qq</b> and <b>pq</b>) for each reaction is denoted in ‘<b>red</b>’, ‘<b>green</b>’ and ‘<b>blue</b>’, respectively. No Call and NPC are denoted in ‘grey’ and NTCs in ‘black’. SNP-GT assay (rs513349) was loaded into sixteen separate assay inlets evenly spaced across the 48.48GT array. The remaining inlets were loaded with a NPC as stated in the Methods section.</p