Finally, we obtained 529,883 clean and high quality sequences for the 10 samples and they were allocated to specific samples according to barcode sequences (Table 1). Table 1 Sample list ID Barcode PCR conditions Read number Chao1 Ace     T* C & E $ (total) (unique) (unique) EPZ-6438 ic50 0.03 (unique) 0.03 A1 TGGAGTAG 1 30 Ex 83,194 17,841 58,148 13,020 108,316 18,590 A2 TGTGACTG 1 30 Ex 158,519 30,361

55,899 34,096 107,984 22,871 B1 CAGACAGA 20 30 Ex 52,793 12,874 39,159 7,455 69,614 9,274 B2 CAGTGAGA 20 30 Ex 78,392 16,846 50,838 8,986 88,268 10,782 C1 CATCTCGT 200 30 Ex 25,705 6,013 16,586 2,700 24,554 2,669 C2 GGTAGGAT 200 30 Ex 25,514 5,968 16,828 2,731 25,294 2,649 D1 GTGTAGAG 20 25 Ex 10,833 3,992 13,749 4,457 26,155 6,406 D2 GTTGGTAC 20 25 Ex 25,181 7,578 22,921 6,698 Selleckchem Tamoxifen 42,784 9,517 E1 GTCAGAGA 20 30 Pfu 34,600 6,750 17,853 6,332 30,589 9,255 E2 GTCTTCTG 20 30 Pfu 35,152 6,818 18,281 6,416 30,434 8,792   Total       529,883 67,826 229,287 34,883 120,750 50,579 *: Dilution folds of the DNA template; &: PCR cycle number; $: Polymerase used (Ex, Ex Taq from Takara; Pfu, PfuUltra II Hotstart 2× Master Mix from Stratagene). Rarefaction analysis We presented the rarefaction curves for OTUs at both unique and 0.03 distances (Fig. 1). The unique OTU represents

both true diversity and PCR very artifacts as described above, while the 0.03 distance OTU may mitigate the effect of PCR mutation artifacts, because the mutation rate in a ~60 bp V6 tag is less than 1 bp (< 3%) [9]. In our present study, we used the nearest distance, rather than the furthest distance, for calculating the OTUs using the Mothur [18]. The reason was that rarefaction curves with different sequencing depth showed consistent trajectory using the nearest distance, but changed with the furthest distance (Additional file 1). Figure 1 Rarefaction curves for the 10 samples using 5 different PCR conditions. A shows the unique (100% similarity) OTU. B

shows 0.03 OTUs at a 97% similarity using the nearest neighbor clustering method. Rarefaction curves for PCR replicates showed consistent trajectories for both unique and 0.03 OTUs (Fig. 1), indicating that the PCR and sequencing steps had good reproducibility. The unique curves for A (1 fold diluted template, 30 cycles), B (20 fold diluted template, 30 cycles) and D (20 fold diluted template, 25 cycles) conditions almost overlapped (Fig. 1A), indicating a similar richness of unique V6 tags in above three conditions. The C condition (200 fold diluted template, 30 cycles) showed a lower slope than the above three, indicating that dilution of DNA template from 20 to 200 fold reduced the V6 diversity of the sample.