3.2- The influence of read sampling depth on taxon
accumulation and α diversity
Read sampling depth strongly influences the number of taxa counted
in an individual PCR replicate (observed alpha diversity). Taxon
accumulation curves (Figure 1) show that for all PCR replicates,
observed α diversity increases with read sampling depth across the range
of depths used. On average across PCR replicates and sites, a read depth
of 1,000 included 51% (PITS) and 33% (FITS) of the observed α
diversity compared to that detected with 20,000 reads. A read depth of
5,000 retained 77% (PITS) and 64% (FITS) of taxa detected at 20,000
reads, and a read depth of 10,000 retained 89% (PITS) and 82% (FITS)
of taxa detected with 20,000 reads. In all sites, the PITS
curves surpass the inflection point where slope begins to decrease
(asymptote) at a sampling depth under 5000 reads. In FITS, the
inflection point is less visually observable and more taxa continue to
accumulate at high read depth (Figure 1). Nonetheless, we observed that
in both PITS and FITS datasets, the maximum observed richness values
vary considerably across PCR replicates.
Because the maximum observed richness could be influenced by read
sampling depth, we explored PCR replicate richness variation using
asymptote values extrapolated to twice the original depth withiNEXT . We found Observed diversity estimates were rarely normally
distributed and variance was high, with up to five replicates per group
being outliers from the mean (Table S3). After outlier removal, PCR
replicate richness at the extrapolated asymptote still exhibited
multiple fold differences in PITS and standard deviations equivalent to
up to 30% of the maximum richness of the group (Table 1). We found the
highest fold differences in observed richness in the PITS data set from
YL.1 (Figure 1e), a site situated within a marine lagoon at a location
that is regularly inundated with both marine water and stream runoff. We
expect these physical processes increase true taxon richness and
possibly heterogeneity within the environmental samples. We observed
fewer outliers in extrapolated richness for FITS, with only up to two
outliers per group, but that variation was high, with standard
deviations up to 21% of the maximum richness of the group. We observed
the highest fold differences in observed richness in FITS at YL.1 and
FO.2.
Increasing the read sampling depth from 1,000 to 10,000 reads resulted
in an average 1.8-fold increase in observed α diversity for PITS and
2.4-fold increase for FITS (Table S4). This increase in observed α
diversity with depth was significant for all six sites and both markers
(Table S4). Shannon diversity did not significantly increase with read
sampling depth for five of the six sites in the PITS data set, but did
significantly increase with all FITS data set (Table S4). Simpson
diversity did not significantly increase with read sampling depth for
any PITS data set, but did for five of the six sites in the FITS data
set (Table S4).