Hauser Philippe M, Bernard Thomas, Greub Gilbert, Jaton Katia, Pagni Marco, Hafen Gaudenz M
Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland.
Vital-IT Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland.
PLoS One. 2014 Mar 5;9(3):e90934. doi: 10.1371/journal.pone.0090934. eCollection 2014.
Determination of the precise composition and variation of microbiota in cystic fibrosis lungs is crucial since chronic inflammation due to microorganisms leads to lung damage and ultimately, death. However, this constitutes a major technical challenge. Culturing of microorganisms does not provide a complete representation of a microbiota, even when using culturomics (high-throughput culture). So far, only PCR-based metagenomics have been investigated. However, these methods are biased towards certain microbial groups, and suffer from uncertain quantification of the different microbial domains. We have explored whole genome sequencing (WGS) using the Illumina high-throughput technology applied directly to DNA extracted from sputa obtained from two cystic fibrosis patients. To detect all microorganism groups, we used four procedures for DNA extraction, each with a different lysis protocol. We avoided biases due to whole DNA amplification thanks to the high efficiency of current Illumina technology. Phylogenomic classification of the reads by three different methods produced similar results. Our results suggest that WGS provides, in a single analysis, a better qualitative and quantitative assessment of microbiota compositions than cultures and PCRs. WGS identified a high quantity of Haemophilus spp. (patient 1) or Staphylococcus spp. plus Streptococcus spp. (patient 2) together with low amounts of anaerobic (Veillonella, Prevotella, Fusobacterium) and aerobic bacteria (Gemella, Moraxella, Granulicatella). WGS suggested that fungal members represented very low proportions of the microbiota, which were detected by cultures and PCRs because of their selectivity. The future increase of reads' sizes and decrease in cost should ensure the usefulness of WGS for the characterisation of microbiota.
确定囊性纤维化肺部微生物群的精确组成和变异至关重要，因为微生物引起的慢性炎症会导致肺部损伤，最终导致死亡。然而，这构成了一项重大的技术挑战。微生物培养并不能完全代表微生物群，即使使用培养组学（高通量培养）也是如此。到目前为止，仅对基于PCR的宏基因组学进行了研究。然而，这些方法偏向于某些微生物群体，并且不同微生物域的定量存在不确定性。我们使用Illumina高通量技术对直接从两名囊性纤维化患者的痰液中提取的DNA进行了全基因组测序（WGS）探索。为了检测所有微生物群体，我们使用了四种DNA提取程序，每种程序都有不同的裂解方案。由于当前Illumina技术的高效性，我们避免了全DNA扩增带来的偏差。通过三种不同方法对读数进行系统发育分类产生了相似的结果。我们的结果表明，与培养和PCR相比，WGS在单次分析中能对微生物群组成进行更好的定性和定量评估。WGS鉴定出大量的嗜血杆菌属（患者1）或葡萄球菌属加链球菌属（患者2），同时还有少量的厌氧菌（韦荣球菌属、普雷沃菌属、梭杆菌属）和好氧菌（孪生球菌属、莫拉克斯菌属、颗粒链菌属）。WGS表明真菌成员在微生物群中所占比例非常低，由于其选择性，通过培养和PCR检测到了它们。未来读数大小的增加和成本的降低应能确保WGS在微生物群特征分析中的实用性。
