For this reason, our Little Penguin monitoring program made the transition to HTS for the 2010 samples. Newly developed HTS platforms, especially small-scale systems such as the GS-Junior or IonTorrent, enable a quick, efficient and relatively inexpensive way to deep-sequence PCR amplicons generated from faecal DNA extracts . Moreover, the use of MID-tagged primers makes it possible to run numerous samples in parallel, enabling not only an overview of the diet composition across a PI3K inhibitor population, but also at the individual level . HTS can provide a wealth of information; greatly increasing the number of DNA sequences returned for a fraction of the labour and associated costs. Concomitant with the increases in sequencing depth is the prospect that HTS data might now provide better quantitative measures of the DNA targets within faecal material, much like estimates obtained using qPCR . In order to compare the quantitative nature of HTS to that of qPCR, a species-specific four fish qPCR assay was designed to estimate the relative abundance of each of the four major prey species determined within the collective samples . Careful development of each of the four primer pairs was critical to data fidelity , as was ensuring that the DNA extracts�� CT values behaved as desired when diluted . From this four fish assay it was clear that H. vittatus and S. sagax were major constituents of the faecal samples; 49% and 32% respectively, with both E. australis and S. robustus each contributing 13% and 5% to the overall composition . The ANF1/ANR2 assay encountered some primer dimer issues at low template copy numbers, however the melt curves enabled differentiation of product and dimer. Although not wholly representative of the total amount of prey DNA within samples, the qPCR assays gave a good indication of the abundance of each of the four major fish species relative to each other. It is GDC-0941 biological activity important to actively compare and contrast both HTS and qPCR approaches to enable an informed decision of the most suitable method to be used for genetic faecal screening. To allow a comparison between both approaches, the HTS data had to be transformed to focus on the same four fish species as the qPCR assay; H. vittatus, S. sagax, E. australis and S. robustus. The proportion of these species to the exclusion of the other species present was determined to be 52%, 32%, 11% and 5% respectively . It is clear that there is a striking degree of similarity between the proportions identified for the four fish species determined by qPCR and HTS . In order to investigate this further, the absolute differences between the results obtained individually by both methods were calculated.