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After several trials testing the mock communities, we decided to use 3000 5000 reads per sample for clustering with a 96% cutoff for the analysis of human gut microbiota.
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Sequencing was carried out on Illumina HiSeq2500 according to the manufacturer's instructions, using 10 pM template per sample for cluster generation, and sequencing kit V2 (Illumina), resulting in ∼40 million paired-end reads per sample.
Notes: *Estimates adjusted for sampling probability and for clustering among patients seen by the same técnico de medicina.
In addition, a feasible size of 96 samples is recommended for clustering to maximize statistical significance.
Theoretical studies (Comas et al. 2011) have also pointed to the worth of using a sampling grid for clustered populations.
Cluster randomised trials need to inflate their sample size to account for clustering.
Randomisation occurred at practice level and an adjustment to the sample size estimates allowed for clustering.
Our study uses SSCC for clustering samples.
Means and SDs of scores are compared using a two-sample t-test, adjusted for clustering.
Consensus clustering has been used for clustering samples to discover and classify cancer types in cancer microarray data [ 1- 4, 6].
These were 'reported accounting for clustering in sample size calculations'reportedrted accounting for clustering in analysis' and 'low or no potential for selection bias'.
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