Amplicon based screening
Sequencing of multiplexed 16S rRNA gene libraries produced high quality
amplicon data. Similar numbers of merged 16S rRNA bacterial sequences
were retrieved across individual Polyplax and Hoplopleurasamples (21, 205 and 20, 201 on average). For the even mock communities
included in the Hoplopleura library as positive controls (see
Methods), the sequencing recovered all 10 bacterial taxa in comparable
abundances. The data from the staggered communities, designed for
testing sequencing sensitivity and PCR bias, confirmed that our approach
can reveal complete microbiome profiles, including low abundant taxa. In
particular, sequences of Bifidobacterium adolescentis (ATCC15703)and Deinococcus radiodurans (ATCC BAA-816) , both present in
0.04% of the original mock DNA template, comprised on average 0.02%
and 0.01% of the reads among the three sequenced mock communities.
However, the staggered composition of the three mock samples did result
in a preferential amplification of one of two dominating taxa, i.e.Staphylococcus epidermidis (ATCC 12228). Compared to the mock
template where this taxon comprises 44.78% of the total DNA, the
average read abundance equaled 70.12%. Therefore, no quantitative
analyses of 16S rRNA gene amplicons were used in this study.