Infectious disease researchers have developed a new approach for the diagnosis of tuberculosis (TB) that relies on shotgun metagenomics, a method for direct sequencing of DNA extracted from sputum samples, which detects and characterizes the Mycobacterium that cause TB without the need for time-consuming culture or enrichment.

Metagenomics is the study of genetic material recovered directly from environmental samples. In metagenomic sequencing, DNA is recovered directly from environmental samples in an untargeted manner with the goal of obtaining an unbiased sample from all genes of all members of the community. Recent studies used shotgun Sanger sequencing or pyrosequencing to recover the sequences of the reads. Shotgun sequencing is a sequencing method designed for analysis of DNA sequences longer than 1,000 base pairs, up to and including entire chromosomes. This method requires the target DNA to be broken into random fragments. After sequencing individual fragments, the sequences can be reassembled on the basis of their overlapping regions

Investigators at Warwick Medical School (United Kingdom) explored the potential of shotgun metagenomics to detect and characterize strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples. To this end, they analyzed eight samples obtained from tuberculosis patients from Gambia.

The concentration of DNA present in each extract was determined using the Qubit (Invitrogen Ltd., Paisley, United Kingdom) 2.0 fluorometer and Qubit dsDNA Assay Kits according to the manufacturer’s protocol using the HS (high-sensitivity) or BR (broad-range) kits, depending on the DNA concentration. There was no detectable DNA in the negative control samples with the HS kit, which is sensitive down to 10 picograms per microliter. DNA extracts were diluted to 0.2 nanograms per microliter and were then converted into sequencing libraries using the Illumina (Little Chesterford, United Kingdom) Nextera XT sample preparation kit. The libraries were sequenced on the Illumina MiSeq instrument at the University of Warwick.

Using this methodology, the investigators were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analyzing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), they were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H, or LAM within the clade of "modern" M. tuberculosis strains.

"Laboratory diagnosis of TB using conventional approaches is a long drawn-out process, which takes weeks or months," said senior author Dr. Mark Pallen, professor of microbial genomics at Warwick Medical School. "Plus, relying on laboratory culture means using techniques that date back to the 1880s! Metagenomics using the latest high-throughput sequencing technologies and some smart bioinformatics, allows us to detect and characterize the bacteria that cause TB in a matter of a day or two, without having to grow the bacteria, while also giving us key insights into their genome sequences and the lineages that they belong to. We have provided proof-of-principle here, but we still need to make metagenomics more sensitive and improve our workflows. But, caveats aside, let us celebrate the fact that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens."

The shotgun metagenomics study was published in the September 23, 2014, online edition of the journal PeerJ.

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