Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow

Genomes can provide information on the ancestry, transmission, and capabilities of a pathogen. For unculturable bacteria, this information can only be obtained through target enrichment. Sequencing bacterial pathogens directly from clinical samples is challenging because of the overwhelming human DNA, the diverse microbiota and extremely low bacteria loads.

In many cases, culture is not an option, due to the sampling method and fastidious bacteria. While applying this technology to sexual transmitted bacteria, we discovered a novel Chlamydia trachomatis LGV lineage called ompA-genotype L4, and confirmed global expansion of the dominant LGV lineage, delivering key insight for molecular diagnostic and epidemiological surveillance. This progress enrichment protocol enables complete genome recovery from positive diagnosed STI clinical samples.

We found that this technique outperforms metagenomic deep sequencing, host DNA depletion, and nanopore adaptive sequencing. We're investigating the circulating strains of these pathogens across Europe and in Argentina. This has the potential to inform regarding transmission and antimicrobial stewardship.

To begin, measure the DNA concentration of the DNA extracts from clinical samples using a sensitive and accurate fluorescence-based method. Dilute each DNA sample using nuclease-free water to 10 to 200 nanograms in a final volume of 17 microliters in each PCR tube strip. Add 3 microliters of fragmentation master mix to each well containing the 17 microliters of diluted DNA.

Pipette up and down 20 times to mix thoroughly. Then, seal the PCR tube strip. Vortex the strip at high speed for 10 seconds and spin briefly to collect the contents.

Then, place the PCR tube strip in the thermal cycler and start the pre-programmed protocol for fragmentation and incubation. Next, prepare the end repair and dA-tailing master mix as shown in the table. Vortex and spin the tube as shown earlier and keep it on ice.

Then, pipette 20 microliters of the end repair and dA-tailing master mix into each well containing 50 microliters of DNA fragments. Seal the strip and vortex at high speed for eight seconds. After briefly spinning the strip, place it back into the thermal cycler.

Once the thermal cycler program finishes, place the PCR tube strip on ice. Next, add 25 microliters of room temperature ligation master mix, which was prepared 30 to 45 minutes in advance to each well containing the DNA fragments. Add 5 microliters of adapter oligo mix for NBC-tagged libraries to each well.

Immediately place the PCR tube strip back into the thermal cycler and continue the program. To begin DNA purification, retrieve the magnetic beads from 4 degrees Celsius and allow them to come to room temperature before use. Then, add 80 microliters to each sample well.

Place the PCR tube strip on the magnetic stand and wash each well twice with 200 microliters of 70%ethanol. Wait for one minute during each wash and carefully remove the ethanol without disturbing the magnetic beads. After mixing and incubating the beads with 35 microliters of nuclease-free water for five minutes, carefully aspirate the cleared supernatant from each well and transfer it into a corresponding well in a new PCR tube strip.

Place the new PCR tube strip on ice and discard the magnetic beads. Take the prepared pre-recapture PCR reaction mix and add 11 microliters of it into each sample well containing the purified DNA library. Add 5 microliters of the selected index prime repair to each reaction and seal the PCR tube strip.

Start the thermal cycler program for PCR amplification as specified in the table to perform the pre-capture PCR. Only add the PCR tube strip once the thermal cycler has reached 98 degrees Celsius. After completing the pre-capture PCR, perform another bead-based cleanup to purify the library.

Incubate the PCR product with magnetic beads. Then, place the PCR strip tube on a magnetic separation device and wait 5 to 10 minutes until the solution becomes clear. While the strip remains on the magnetic stand, carefully remove and discard the clear supernatant from each well.

Then, elute the DNA in 15 microliters of nuclease-free water and measure the DNA concentration of the purified DNA library. For sample hybridization, program the thermal cycler according to the protocol specified here. Start the program and immediately pause the run.

Dilute 500 to 1, 000 nanograms of the prepared DNA libraries to a final volume of 12 microliters using nuclease-free water. After vortexing and spinning down the oligonucleotide blocker mix, add 5 microliters to each sample tube. Seal the PCR tube strip and place it in the thermal cycler.

Pause the thermal cycler when it reaches step three of the protocol. Add 13 microliters of the prepared room temperature probe hybridization mix to each sample while keeping the strip inside the thermal cycler. Pipette slowly up and down 8 to 10 times to mix.

Ensure no air bubbles are present, and place the strip back into the thermal cycler. Then, confirm that all tubes are sealed tightly. To capture the hybridized library, transfer the entire volume from each tube to the corresponding tubes containing 200 microliters of pre-washed streptavidin beads.

Collect the streptavidin beads before washing them with room temperature wash buffer. Once the PCR tube strip is removed from the magnetic separator, add 200 microliters of high temperature wash buffer pre-warmed to 70 degrees Celsius and re-suspend the beads by pipetting up and down. Seal the tube strip, vortex at high speed, and spin quickly as demonstrated earlier.

Place the sealed tube strip in the thermal cycler block at 70 degrees Celsius and incubate for 5 minutes. Transfer the PCR tube strip from the thermal cycler back to the magnetic separator at room temperature. Wait for one minute until the solution becomes clear.

Then, carefully remove and discard the supernatant. Then, add 25 microliters of nuclease-free water to each tube containing the beads. Program the thermal cycler as specified in the table shown here.

Add 25 microliters of the prepared post-capture PCR reaction mix to each sample tube. Mix the reaction until the suspension is uniform, and securely seal the PCR tube strip without spinning it down. Place the sealed PCR tube strip in the thermal cycler.

After transferring the PCR product to a fresh tube, add 50 microliters of bead suspension to each sample tube and perform the DNA cleanup as shown earlier. Measure the DNA concentration of the final purified library DNA using a fluorescence-based quantification method. Target enrichment resulted in significantly higher percentages of on-target reads, and therefore complete genomes compared to direct sequencing across all pathogens tested, including Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma Genitalium.

Genome sequencing success was predominantly observed in samples with qPCR cycle threshold values below 30 for all four pathogens. With a clear inverse correlation between CT values and percentage of on-target reads for Chlamydia trachomatis. Using a hybridization temperature of 62.5 degrees Celsius compared to the previous 65 degrees Celsius, improved genome coverage for mycoplasma genitalium without reducing performance for the other target pathogens.

Phylogenetic analysis of Chlamydia trachomatis genomes from Argentina showed many samples clustering within the L2b clade and revealed a novel lineage proposed as ompA-genotype L4 separated from all previous LGV genomes by approximately 600 single nucleotide polymorphisms.