Abstract
Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.
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Supplementary Table S1
Taxonomy assignments of 16S rDNA cloned sequences. Taxonomy assignments for each 16S rDNA cloned sequence was performed using the GenBank database by selecting the nearest neighbour (highest bit score) from the BLAST results. (JPEG 42 kb)
Supplementary Table S2
Read distribution at the species level. Partial 16S rDNA sequences in the RDP-II database are in bold. The field ‘Hit count’ displays the number of reads with the species in ‘Hit Description’ as their nearest neighbour (similarity searching). Only species with more than 20 BLAST hits are shown. (JPEG 136 kb)
Supplementary Table S3
Taxonomic assignments for the 16S rDNA assembled sequences. SI and SC are the sequence identity and sequence coverage, respectively. Taxonomy assignment of each 16S rDNA assembled sequence is performed using the RDP-II database and selecting the nearest neighbour (highest bit score) from the BLAST results. (JPEG 51 kb)
Supplementary Table S4
Identification of the Rp2 primer sequence at the beginning of 16S rDNA genes. SI and SC are the sequence identity and sequence coverage, respectively. The Rp2 primer sequence was BLASTed against the entire RDP-II database. BLAST results with perfect sequence identity and coverage were filtered according to the position of the Rp2 primer in the sequence. Species matching the Rp2 primer in the first 100 base pairs (bp) of their 16S rDNA sequence are displayed. (JPEG 120 kb)
Supplementary Figure S1
Read distribution at the genus taxonomic level. Classification of the pyrosequencing reads using the RDP-II classifier tool and BLAST search. (JPEG 62 kb)
Supplementary Figure S2
Read coverage of A. marinus 16S rDNA sequence. (JPEG 70 kb)
Supplementary Figure S3
Read coverage of the 16S rDNA assembled sequences. Read coverage of the 16S rDNA assembled sequences of five species with a sequence identity >98.5% and a sequence coverage equal to 100%. The red bars under the x-axis define the hypervariable regions. (JPEG 119 kb)
Supplementary Figure S4
Repartitioning of the remaining reads. Classification of the remaining reads using BLAST searching with a sequence identity ≥97% and a sequence coverage >80%. Genus groups or species with more than 20 reads are shown. (JPEG 66 kb)
Supplementary Figure S5
Read coverage of the uncultured Gemella sp. 16S rDNA gene. The red bars under the x-axis define the hypervariable regions. (JPEG 73 kb)
Supplementary Figure S6
Read length distribution of the pyrosequencing data. (JPEG 56 kb)
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Armougom, F., Bittar, F., Stremler, N. et al. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing. Eur J Clin Microbiol Infect Dis 28, 1151–1154 (2009). https://doi.org/10.1007/s10096-009-0749-x
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DOI: https://doi.org/10.1007/s10096-009-0749-x