Article Figures & Data
Figures
- FIG. 1.
Schematic of normalization methods. PCR amplicons were generated for 144 different bar codes and processed for normalization and pooling by the three methods shown schematically. The three methods were direct quantification (NanoDrop), size-restricted DNA quantification (Qiagen), and (iii) quantitative DNA binding (SequalPrep kit; Invitrogen). A single pool of PCR product was split and processed by the three normalization methods. Individual amplicons were purified with the Ampure magnetic bead purification kit prior to determination of DNA concentration. †, the Qiagen pool was purified with the same kit prior to sequencing. ‡, the Invitrogen pool was concentrated using the Zymo DNA Clean & Concentrator kit prior to sequencing. Spec., specimen.
- FIG. 2.
Coefficient of variation from triplicate samples by the three methods. The three methods were direct quantification (NanoDrop), size-restricted DNA quantification (Qiagen), and (iii) quantitative DNA binding (Invitrogen). The coefficient of variation calculated from sequence counts for samples amplified with three independent bar codes (n = 44) were calculated to show how well the sequence counts agreed. A lower CV represents tighter clustering of the counts.
Tables
- TABLE 1.
Counting statistics for each amplicon pool
Counting statistic Value for pool constructed by methoda: Invitrogen NanoDrop Qiagen No. of sequences Total 52,896 29,353 39,372 Minimum 195 0 21 Maximum 629 553 1,584 Avg 367.3 203.8 273.4 SD 79.8 107.9 206.7 Maximum/minimum ratio 3.2 >553b 75.4 Failure rate (%) 2.1 43.8 62.5 ↵ a Three methods were used to construct the amplicon pools: (i) direct quantification (NanoDrop 1000; NanoDrop), (ii) size-restricted DNA quantification (QIAxcel; Qiagen), and (iii) quantitative DNA binding (SequalPrep kit; Invitrogen).
↵ b The minimum was set at 1 for the purpose of this calculation.
- TABLE 2.
Processing time for each step of pool construction
a Primer removal was performed on a single aliquot of the pooled amplicons by the same procedure used for the individual amplicons in the NanoDrop pool.
b This represents manual adjustment of the output for each sample. Software modification would shorten this time significantly.
- TABLE 3.
Microbial richness estimation for each DNA sample
DNA sample Sanger sequencing % Chao estimate observed (Sanger/ pyrosequencing)b Pyrosequencing No. of sequences No. of OTUs Diversity estimate using Chao1 % Chao estimate observeda No. of sequences No. of OTUs Diversity estimate using Chao1 % Chao estimate observedc 1 75 38 88 43.18 2.30 1,761 655 1,654 39.60 2 96 69 289 23.88 3.27 1,818 873 2,107 41.43 3 94 17 45 37.78 0.64 2,338 1,185 2,674 44.32 4 60 64 180 35.56 4.77 1,303 616 1,342 45.90 5 37 36 333.5 10.79 1.38 1,956 992 2,613 37.96 6 74 53 139.7 37.94 3.09 2,120 775 1,714 45.22 7 92 11 39 28.21 2.59 2,109 192 424 45.28 8 89 60 273 21.98 2.26 3,680 1,209 2,659 45.47 9 93 55 213 25.82 1.82 1,898 1,129 3,025 37.32 10 80 58 271 21.40 2.53 2,119 1,056 2,295 46.01 11 85 53 140 37.86 1.28 3,948 1,753 4,149 42.25 12 62 36 148 24.32 1.58 2,705 901 2,280 39.52 13 73 66 361 18.28 2.21 1,459 923 2,991 30.86 14 90 25 55 45.45 1.39 2,375 519 1,795 28.91 15 68 54 178 30.34 2.34 2,689 960 2,312 41.52 16 81 55 228 24.12 7.71 3,150 324 713 45.44 17 92 21 51 41.18 22.93 540 46 91.6 50.22 18 85 20 48 41.67 13.33 505 89 150 59.33
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Sequence counts from each amplicon library analyzed (Table S1).
PDF file, 28K.
- Supplemental file 1 -
Sequence counts from each amplicon library analyzed (Table S1).
