Table 1.
Details for the experiments conducted.
Fig 1.
Definitions used in assessing the importance of analysis parameters.
Shown are the definitions for quality and abundance filtering methods used in assessing their impact on both the number of operational taxonomic units (OTUs) and distance-based operational taxonomic units (DTUs) [24] obtained for a given sample. maxee—Maximum Expected Error
Fig 2.
Quantitative PCR and sequencing results of the sample screening assay.
Quantitative PCR curves indicating the presence of DNA and the degree of inhibition (LEFT) with agarose gel electrophoresis clearly indicating the presence of DNA post amplification via means of strong bands (INSET ON GRAPH). Samples were subsequently sequenced and the percentage abundance of two fish genera is indicated, where, based on taxa-specific quantitative PCR results, Sardinops (specifically S. sagax—Australian pilchard) should be in the highest abundance, with Engraulis (specifically E. australis—Australian anchovy) being in the lowest abundance. (RIGHT).
Fig 3.
Average sequencing error rates across a single amplicon region.
Average sequencing error rates are shown for multiple bird species across the whole of a short 12S rRNA gene region (A). Additionally, the error profile across the gene region is shown for Calyptorhynchus lathami for both the Ion Torrent PGM (B) and MiSeq (C) with key. The error patterns observed were similar across all species sequenced. Error rates are shown across 5 bp segments and where error rates were above 1% for a single base this is indicated through the red circles.
Fig 4.
Impact of analysis parameters on the numbers of taxonomic units obtained for a bulk-bone sample.
A number of analysis parameters were used to analyse a complex mixture containing numerous taxa. Different quality and abundance filtering methods were used in addition to two taxonomy-independent measures of analysis, full definitions and explanations of which are in Fig 1. The spread in the numbers of taxonomic units obtained across the combinations of parameters chosen is seen. The radius of each semicircle represents the number of taxonomic units obtained given a set combination of the parameters used. The number of taxonomic units is also indicated above each semicircle. Each semicircle is proportional to all others. AFM—abundance filtering method; QFM—quality filtering method; TIM—taxonomy-independent method.