ABSTRACT
The lung microbiome has been shown to reflect a range of pulmonary diseases-for example: asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Studies have now begun to show microbiological changes in the lung that correlate with lung cancer (LC) which could provide new insights into lung carcinogenesis and new biomarkers for disease screening. Clinical studies have suggested that infections with tuberculosis or pneumonia increased the risk of LC possibly through inflammatory or immunological changes. These have now been superseded by genomic-based microbiome sequencing studies based on bronchoalveolar lavage, sputum or saliva samples. Although some discrepancies exist, many have suggested changes in particular bacterial genera in LC samples particularly, Granulicatella, Streptococcus and Veillonella. Granulicatella is of particular interest, as it appeared to show LC stage-specific increases in abundance. We propose that these microbial community changes are likely to reflect biochemical changes in the LC lung, linked to an increase in anaerobic environmental niches and altered pyridoxal/polyamine/nitrogenous metabolism to which Granulicatella could be particularly responsive. These are clearly preliminary observations and many more expansive studies are required to develop our understanding of the LC microbiome.
ABSTRACT
BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.
