Article Figures & Data
Figures
- Fig 1
Design of dual-index sequencing strategy and schematic describing the four sequencing reads. The primers specific to the 16S rRNA gene are shown in boldface black text, linkers are in blue, pads are in green, the index region is in red, and the adapters are underlined. This schematic is demonstrated using the V4-specific primer sequences and linkers. The PCR and sequencing primers for each of the three regions are provided in the supplemental material.
- Fig 2
Profile of sequencing errors in the first and second read (A and C) and the quality scores associated with different types of errors in the first and second read (B and D) using data from run 130403.
- Fig 3
Relationship between the error rate and the fraction of sequences kept as a function of the ΔQ value for the V34, V4, and V45 regions using data from run 130403.
- Fig 4
Principal coordinate ordination of ϴYC values (28) relating the community structures of the fecal microbiota from 12 mice collected on days 0 through 9 (Early) and days 141 through 150 (Late) after weaning.
Tables
- Table 1
Summary of operating conditions for various MiSeq sequencing runs and machine-reported quality metricsa
Run [DNA] (pM) Cluster density (103/mm2) % PhiX % of bases ≥Q30b Reported machine error (%) No. of 16S rRNA pairs (×106) 130401 10.0 1,313 3.8 63.7 1.31 12.6 130403 5.0 1,094 3.2 70.5 0.89 12.4 130417 3.0 839 6.2 74.6 0.92 10.5 130422 1.8 690 8.0 80.1 0.65 9.0 - Table 2
Summary of the error rates and number of observed OTUs for the sequencing runs described in Table 1
Region and run Error rate (%) for: % reads remaining from basic (ΔQ = 0) Average no. of OTUsa Basic ΔQ = 6 Precluster Mockb Mockc Soil Mouse Human V34 130401 2.14 0.37 0.21 10.3 26.9 49.6 1,110.6 175.1 187.5 130403 1.30 0.26 0.12 27.6 31.1 47.8 1,095.8 158.2 164.1 130417 1.12 0.24 0.10 27.9 35.1 52.2 1,038.6 ND 142.6 130422 0.91 0.29 0.17 47.5 41.4 54.3 1,053.0 162.5 145.1 V4 130401 1.08 0.06 0.01 44.2 23.5 43.4 1,248.2 136.1 115.4 130403 0.67 0.05 0.01 60.1 23.5 40.9 1,261.8 133.9 117.8 130417 0.40 0.05 0.01 69.3 22.8 37.5 1,257.3 135.5 117.2 130422 0.28 0.05 0.01 78.4 23.2 37.2 1,256.2 132.8 117.3 V45 130401 4.60 0.87 0.64 13.5 191.9 271.7 1,462.8 198.0 312.9 130403 3.31 0.79 0.56 32.3 180.4 246.1 1,519.7 213.3 324.0 130417 2.38 0.66 0.43 36.5 110.8 158.3 ND 180.4 242.1 130422 1.67 0.58 0.36 56.4 98.0 131.6 1,403.3 186.3 227.2 ↵a The average number of OTUs is based on rarefaction of each sample to 5,000 sequences per sample; cells labeled ND reflect samples that did not have at least one replicate with more than 5,000 sequences.
↵b Number of OTUs in the mock community when all chimeras were removed; in the absence of chimeras and sequencing errors, there should be 20 OTUs for all three regions.
↵c Number of OTUs in the mock community when chimeras were removed using UCHIME.
- Table 3
Summary of assemblies using shotgun sequence data generated in parallel to 16S rRNA gene sequences
Library No. of reads (×106) No. of bases (×106 bp) No. of contigs (≥500 bp each) N50a (×103 bp) Reads that mapped to contigs (%) Clostridium clostridioforme D4 1.44 360 317 37.88 97 C. clostridioforme CIP110249 1.54 380 323 43.80 96 Mock community 2.40 600 31,946 1.46 66 Human feces 5.79 1,450 27,321 1.00 49 ↵a The contig length where all contigs of that length or longer contain more than 50% of the bases found across all contigs.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Challenges of sequencing the 16S rRNA gene with the MiSeq Platform, summary of operating conditions for various MiSeq sequencing runs and machine reported quality metrics (Table S1), summary of the error rates and number of observed OTUs for the sequencing runs described in Table S1 (Table S2), and protocols.
PDF, 996K
- Supplemental file 1 -
