Using a combination of immunomagnetic separation and multiple displacement amplification, or IMS-MDA, a team led by researchers at the Wellcome Trust Sanger Institute has developed a method allowing sequencing of whole genomes from discarded molecular diagnostics samples, without the need for in vitro culture.
The group has applied the technique initially to Chlamydia trachomatis with the aim of conducting a global genomic survey of the difficult-to-culture bacteria to better understand its evolution and effect on public health. The researchers published a description of the method and its development in Genome Research last month.
Nicholas Thomson, the paper's senior author, told In Sequence that to understand how chlamydia spreads, researchers need an understanding of the genomic diversity of the bacteria, especially those strains that circulate and infect people in the real world. But difficulties in culturing the pathogen and the cessation of culturing as a diagnostic tool have made sequencing a large number of samples too difficult and costly.
"Before about the year 2000 most countries' gold standard for diagnosis was culture – so you could get hold of cultured samples. But now most health services have stopped doing that and are doing molecular tests to diagnose chlamydia," Thomson said.
"It's entirely impractical [for us] to grow 400 or more strains — or however many we need to do a global genomic survey — but we recognized that there is a huge availability of discarded clinical diagnostic samples," he added. "So we decided that because most sexual health clinics and surveillance labs were not going to culture any more we would develop a way of pulling bacteria straight out of a diagnostic sample, and then using established techniques we'd amplify the whole genome, sequence that, and use that to do our global survey."
In the study, Thomson and his colleagues described their method, which uses immunomagnetic separation to enrich and extract bacterial DNA from mixed clinical diagnostic samples. For Chlamydia, the group used a commercially-available mouse antibody against the bacteria to separate the bacteria in the samples. A second anti-mouse antibody attached to magnetic beads allowed the researchers to then draw the bound bacteria out.
The group then used multiple displacement amplification to amplify the Chlamydia DNA to a sufficient level for sequencing, and sequenced the samples using standard Illumina protocols on either a GAII or HiSeq.
To assess the method's ability to assemble high-quality and complete genomes, the researchers evaluated how different samples' DNA concentrations affected the depth of coverage they were able to achieve.
Overall, the group was able to generate complete genome sequences for five out of 18 clinical samples subjected to the method — an approximately 20 percent success rate. According to the team, for the five samples that resulted in reads covering more than 99 percent of the Chlamydia chromosome and a mean depth of coverage of more than 38X, each contained at least 10,0000,000 genome copies per microliter after the IMS-MDA procedure.
While the method can't generate a complete genome from every clinical sample, especially those with lower concentrations of DNA available, Thomson said that his team is excited about the technique. "The genesis of the idea to actually do this was that there was no other way of getting these sequences," he said. "This really was the only approach we could do to get whole genomes from clinical samples."
In addition to testing the method as a way of generating whole genomes to examine genome variation, the group also examined whether the five clinical samples they sequenced could also be used to produce de novo genome assemblies.
The authors wrote that sequence data generated using MDA has more uneven depth of coverage than what you can get from cultured samples. As a result, genomes built using the IMS-MDA method should be problematic for assembly algorithms. However, the group was able to use the SPAdes algorithm to assemble de novo genomes for the five samples that were consistent with the expected appropriate chromosome size and to derive genotypes for the five strains.
Thomson said the group was also able to resolve mixed infections in the study as long as there was sufficient bias between strains
"You're always sampling a population that you hope is not a mixed population. But we showed that even if it is a mixed population you can still resolve those sequences, although there will be a point after which you couldn’t," he said.
According to the study authors, the full IMS-MDA protocol can be performed within five hours with little hands-on time. The team also showed that it could be used on Chlamydia samples that were archived and stored for as long as several years.
Thomson said the group chose to use Illumina because it was looking for a high depth of coverage. "The more coverage you have, the more accurate it is and the more possibility you have to resolve multiple infections," he explained.
But, he said, depending on what questions a team is looking to answer, the IMS-MDA method could work as a preparation step before sequencing using any sequencing technology.
Thomson and his Sanger team are planning to continue to use the approach to follow Chlamydia populations, "getting genomes for as diverse a set of bacteria as we can," he said, "to understand if there are differences between strains causing different diseases.
"Clearly [clinical samples] are what contain strains that are actually causing disease, and so are where we might find novel insights," he said.
Additionally, the team is interested in using the method to look at complex environmental samples of diarrhea-causing pathogens like Shigella.
"If you have a specific antibody that recognizes those bacteria, then this will work, and there are commercial antibodies available for most bacteria, so this technique should work for all kinds of targets," Thomson said.