3.3 Within-site variation composition and relative
abundance (RA)
We next explored similarity between PCR replicates by comparing the
RA of taxa detected in them. Qualitatively, for both PITS and FITS,
within-site PCR replicates appear similar to each other and each site is
readily distinguishable from other sites (Figure 3). Only one replicate
appeared to have a different community profile, and this was in PITS
results from the YL.1 site (Figure 3). Analysis of ‘local contribution
to beta diversity’ (LCBD) provides a quantitative examination of
differences in composition and RA. At a read sampling depth of 5,000 and
with a five read minimum cutoff, LCBD statistics identified several PCR
outliers in the PITS data set (11 PCR replicates from the YL.1 site and
2 PCR replicates from the FO.2 site; Table S5) and one outlier in the
FITS data set (1 outlier replicate from the FO.2 data set; Table S5).
To explore how read sampling depth and minimum read cutoff influence
LCBD, we repeated these analyses at all three read sampling depths
(1,000, 5,000, and 10,000 reads) with minimum read cutoffs of two, five,
or ten reads (Table S5), and performed Chi-squared tests for significant
differences among groups. The number of LCBD-based outliers increased
significantly with higher read sampling depth in the FITS data set (p=
3.861E-15), but not in the PITS data set (p=0.25). We found no
significant effect of minimum read cutoff for either the PITS (p=0.71)
or FITS (p=0. 79) data set, suggesting that low abundance taxa, which
are most likely to be impacted by changing the minimum read cutoff, are
not causing outliers. For both the PITS and FITS data set, we found that
site itself has a significant effect on the number of observed PCR
outliers (both p <2.2e-16).