Effect of sequence depth on intra-sample divergence.

<p>Divergence is expressed as (A) Bray-Curtis and (B) Jaccard dissimilarity between repeated (i = 100) random sub-samples of all replicates with >15,000 reads. Error bars represent bootstrapped standard errors.</p

Effect of sequence depth per replicate on mean silhouette score, representing the separability of replicates based on sample of origin, based on repeated sub-sampling of OTUs.

<p>Filtering to protist or metazoan OTUs was carried out after sub-sampling. Error bars represent bootstrapped standard errors.</p

Expected OTU richness as a function of sequence depth (rarefaction).

<p><i>In silico</i> pooled replicates are shown as dashed lines and individual extraction replicates as solid lines. In panel A richness up to the maximum read number for pooled replicates and in B the same rarefaction curves up to maximum read number for individual replicates. Error bars (in B) represent standard error, for pooled samples calculated as described in Heck et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179443#pone.0179443.ref047" target="_blank">47</a>].</p

Effect of pooled extraction replicate number on OTU richness and Shannon diversity.

<p>Mean diversity estimates based on repeated (i = 100) random selection of replicates to pool followed by random sub-sampling of OTUs to a total sequence depth of 15,000 reads per sample (divided equally between selected replicates). Filtering to protist or metazoan OTUs was carried out after sub-sampling. Error bars represent the standard error of values from the bootstrapping procedure described above.</p

Sample processing including technical replication of DNA extraction and amplicon library preparation.

<p>Sample processing including technical replication of DNA extraction and amplicon library preparation.</p

OTU_matrix_99_similarity

A presence absence matrix for each OTU and specimen using 99% similarity to assign OTUs. See read me file in 'OTU_matrix_97_similarity.tab

Effect of pooled extraction replicate number on silhouette score.

<p>Mean silhouette scores were calculated based on repeated random selection of replicates to pool and sub-sampling of OTUs to a total sequence depth of 15,000 reads per sample (divided equally between selected replicates). Filtering to protist or metazoan OTUs was carried out after sub-sampling. Error bars represent bootstrapped standard errors.</p

OTU_best_rep_99_similarity

Best representative sequences from each OTU based on 99% similarity

Rarefaction comparing expected OTU richness of pooled samples (grey lines) and individual replicates (black lines).

<p>Only replicates with more than 15,000 reads were included from the samples Fine Sand (A; n = 5), Coarse Sand (B; n = 7) and Clay (C, n = 5). Panel D shows expected richness in pooled samples compared to mean expected replicate richness. Error bars represent standard error, for pooled samples calculated as described in Heck et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179443#pone.0179443.ref047" target="_blank">47</a>].</p

OTU_matrix_98_similarity

A presence absence matrix for each OTU and specimen using 98% similarity to assign OTUs. See read me file in 'OTU_matrix_97_similarity.tab