Of these sequences ,93% were unambiguously attributed to eleven fish species and ,0.1% were identified as belonging to the genus Pelates . There were low levels of human contamination and penguin DNA and unassigned/uninformative sequences accounted for ,3.6% of sequences. There was notable variation in the AP24534 structure number of sequences LY294002 cost generated for each faecal sample , and this is likely due to inaccurate blending of amplicons . However, an average of ,300 reads per sample is more than sufficient coverage for dietary audits, especially when compared to the average number of sequences often generated per sample using bacterial cloning . HTS of the Oct ��10-Dec ��10 samples revealed that, of the prey items identified, H. vittatus, S. sagax, E. australis and S. robustus were the major species present within the faecal material, each contributing 49%, 32%, 11% and 5% respectively . The remaining fish identified were minor contributors to the overall composition of the samples and only in one sample did any of these fish constitute a significant proportion of the prey detected, that of PEN_42, where Parequula melbournensis contributed 48% to the sample composition for this individual . It is clear from the bacterial cloning and HTS data that there were four dominant fish species detected within the samples at this study site, those being H. vittatus, S. sagax, E. australis and S. robustus . The occurrence of other minor contributing prey items within the samples is consistent with previous findings and reflects the opportunistic feeding behaviour of the Little Penguins . A direct comparison of cloning and HTS is somewhat hampered by the fact that different faecal samples from different time periods were used for each method. However, it is clear that a number of important conclusions can be drawn from both datasets. Both methods provide a clear picture of the major prey species that are present within the collective faecal samples. Where they differ is in the relative contribution of each of these individual species , however this could be a result of temporal effects as it is well documented that the diet of Little Penguins varies throughout the year . Cloning of universally amplified PCR products using bacteria, followed by DNA purification and Sanger sequencing is both expensive and time consuming. An additional issue, not entirely observed in this study, is that large numbers of clones are required in order to detect rare species , with the associated time and expense being inefficient for long-term monitoring of species diet.