Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA. Results We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge. Conclusion We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites

Perbedaan Persepsi Risiko Audit, Materialitas dan Kualitas Audit Sebelum dan Sesudah Implementasi Ketentuan Pidana UU No. 5 Tahun 2011 Tentang Akuntan Publik (Studi Persepsi pada Kantor Akuntan Publik Surabaya)

Penelitian ini bertujuan untuk memperoleh data informasi dalam hal bukti empiris tentang perbedaan persepsiauditor dalam menentukan risiko audit, tingkat materialitas dan kualitas audit sebelum dan sesudahpelaksanaan ketentuan pidana UU No 5 tahun 2011 tentang akuntan publik.Populasi dalam penelitian ini adalah auditor yang bekerja pada Kantor Akuntan Publik (PAF) di Surabayasebagai 46 PAF. Auditor dipilih sebagai sampel dalam penelitian ini adalah auditor yang menjabat sebagaipartner, manajer, pengawas dan senior. Data yang digunakan dalam penelitian ini adalah data primer dengankuesioner yang dibagikan sebanyak 236 kuesioner. Metode analisis yang digunakan dalam penelitian iniadalah uji beda dengan menggunakan paired sample t-test.Hasil penelitian ini menunjukkan ada perbedaan persepsi auditor dalam menentukan risiko audit, tingkatmaterialitas dan kualitas audit sebelum dan sesudah pelaksanaan ketentuan pidana UU No 5 tahun 2011tentang akuntan publik.Keyword : Risiko audit, Materialitas, kualitas Audit, ketentuan pidana dan UU Nomor 5 tahun 2011 tentangakuntan publik

Influence of composite particle formation on the performance and economics of grit removal

Grit is routinely removed at the headworks of municipal wastewater treatment works to limit its onerous impact on downstream processes. Grit separation technologies are normally based on sedimentation of a homogeneous material (usually sand). However, in practice inorganic grit particles are likely to be combined with organic matter, such as fats oils and grease (FOG), producing a composite particle whose settling properties vary with the inorganic/organic content. A study of the impact of particle composition on its sedimentation has been conducted encompassing theoretical description (for particle settling in transitional flow), practical measurement and economic analysis. Practical measurement included sedimentation tests of homogeneous and composite particles along with characterisation of accumulated granular material sampled from actual municipal wastewater treatment works. The economic assessment was based on data from full-scale installations in the UK and US pertaining to remedial measures undertaken as a result of grit impacts, primarily accumulation in vessels and channels and damage of mechanical equipment through abrasion. Practical tests revealed coating of the sand grains with a FOG analogue (candlewax) to generate composite particles containing 45% wax by weight. The coated particles were then 30% less dense, 22% larger and 14% less settleable, on average, than the uncoated particles. Samples of accumulated grit taken from anaerobic digesters and aeration lanes from a full-scale plant indicated a FOG content (43%) similar to that of the waxed particles in the bench-scale tests, thus leading to a similar grain retardation of 14% assuming the FOG to be entirely associated with the grit. An assessment of the impact of the consequential breakthrough of grit particles due to buoyancy generated by composite particle formation indicated a $1.1 increase in operating costs per megalitre (ML) wastewater

The Status of the Rufous Scrub-Bird 'Atrichornis Rufescens': Habitat, Geographical Variation and Abundance

The status of the Rufous Scrub-bird 'Atrichornis rufescens' has always been difficult to assess due to the bird's secretive nature and the density of its habitat. The primary aim of this study has been to provide a scientific basis for future assessment of status by answering questions concerning three aspects of the species' biology: detectability, habitat requirements, and geographical variation. A secondary aim of the study has been to provide a preliminary assessment of status based on the results of an exploratory survey, interpreted within the context of the intensive research findings

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

Nonlethal age estimation of three threatened fish species using DNA methylation: Australian lungfish, Murray cod and Mary River cod

Age-based demography is fundamental to management of wild fish populations. Age estimates for individuals can determine rates of change in key life-history parameters such as length, maturity, mortality and fecundity. These age-based characteristics are critical for population viability analysis in endangered species and for developing sustainable harvest strategies. For teleost fish, age has traditionally been determined by counting increments formed in calcified structures such as otoliths. However, the collection of otoliths is lethal and therefore undesirable for threatened species. At a molecular level, age can be predicted by measuring DNA methylation. Here, we use previously identified age-associated sites of DNA methylation in zebrafish (Danio rerio) to develop two epigenetic clocks for three threatened freshwater fish species. One epigenetic clock was developed for the Australian lungfish (Neoceratodus forsteri) and the second for the Murray cod (Maccullochella peelii) and Mary River cod (Maccullochella mariensis). Age estimation models were calibrated using either known-age individuals, ages derived from otoliths or bomb radiocarbon dating of scales. We demonstrate a high Pearson's correlation between the chronological and predicted age in both the Lungfish clock (cor = .98) and Maccullochella clock (cor = .92). The median absolute error rate for both epigenetic clocks was also low (Lungfish = 0.86 years; Maccullochella = 0.34 years). This study demonstrates the transferability of DNA methylation sites for age prediction between highly phylogenetically divergent fish species. Given the method is nonlethal and suited to automation, age prediction by DNA methylation has the potential to improve fisheries and other wildlife management settings

DNA metabarcoding as a marine conservation and management tool: A circumpolar examination of fishery discards in the diet of threatened albatrosses

Almost all of the world's fisheries overlap spatially and temporally with foraging seabirds, with impacts that range from food supplementation (through scavenging behind vessels), to resource competition and incidental mortality. The nature and extent of interactions between seabirds and fisheries vary, as does the level and efficacy of management and mitigation. Seabird dietary studies provide information on prey diversity and often identify species that are also caught in fisheries, providing evidence of linkages which can be used to improve ecosystem based management of fisheries. However, species identification of fish can be difficult with conventional dietary techniques. The black-browed albatross (Thalassarche melanophris) has a circumpolar distribution and has suffered major population declines due primarily to incidental mortality in fisheries. We use DNA metabarcoding of black-browed albatross scats to investigate their fish prey during the breeding season at six sites across their range, over two seasons. We identify the spatial and temporal diversity of fish in their diets and overlaps with fisheries operating in adjacent waters. Across all sites, 51 fish species from 33 families were identified, with 23 species contributing >10% of the proportion of samples or sequences at any site. There was extensive geographic variation but little inter-annual variability in fish species consumed. Several fish species that are not easily accessible to albatross, but are commercially harvested or by-caught, were detected in the albatross diet during the breeding season. This was particularly evident at the Falkland Islands and Iles Kerguelen where higher fishery catch amounts (or discard amounts where known) corresponded to higher occurrence of these species in diet samples. This study indicates ongoing interactions with fisheries through consumption of fishery discards, increasing the risk of seabird mortality. Breeding success was higher at sites where fisheries discards were detected in the diet, highlighting the need to minimize discarding to reduce impacts on the ecosystem. DNA metabarcoding provides a valuable non-invasive tool for assessing the fish prey of seabirds across broad geographic ranges. This provides an avenue for fishery resource managers to assess compliance of fisheries with discard policies and the level of interaction with scavenging seabirds

Sex identification from distinctive gene expression patterns in Antarctic krill (Euphausia superba)

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