The overall goal of this study is to implement whole genome sequencing for identification of mutations in genes that confer antifungal drug resistance in Candida glabrata in a diagnostic laboratory. Whole genome sequencing of fungi can make significant contributions in the field of antifungal drug resistance. It allows simultaneous detection of genome-wide mutations in drug-resistant fungi to important antifungal agents.
Whole genome sequencing of medically important fungi can also enable the detection and monitoring of outbreaks, of fungal disease, and transmission events in health care settings, as well as emergence of fungal species and clones that clinicians would need to know about. Demonstrating this procedure will be Dr.Rebecca Rockett and Dr.Verlaine Timms both doctoral research fellows from our laboratory. To begin, prepare genomic DNA samples from clinical and commercial strains of Candida glabrata.
Then add five microliters of quantified DNA to each well of previously-labeled fresh 96-well hard-shell thin-wall plate. Add 10 microliters of tagmentation buffer and five microliters of amplification buffer to each well. Then use a pipette to gently mix the samples and seal the plate with an adhesive seal, put the plate inside a thermal cycler, and run the appropriate PCR program.
When the sample has reached 10 degrees Celsius, immediately add five microliters of neutralization buffer and use a pipette to gently mix the samples. Then seal the plate, and incubate at room temperature for five minutes. After the incubation period, add 15 microliters of Indexing PCR Master Mix to each well.
Arrange primer tools in an index plate rack and record the position of the indices on the template. Then place the tagmentation plate containing PCR Master Mix on the index plate rack with the index tubes in the correct order. It is important to arrange the index primer one tubes in horizontal orientation and the index primer two tubes in vertical orientation.
It is based on the order and position provided in the index template. Remove the old caps from the index primer tubes and discard them. With a multi-channel pipette, add five microliters of index primers to each well.
It is important to perform this step carefully in order to avoid cross contamination between samples and indices. To prevent cross contamination between indices, replace each of the old index tube caps. Finally, seal the plate and run the second PCR program.
First transfer the PCR product from the tagmentation plate to a deep-well plate. Vortex a magnetic bead solution and add 30 microliters of beads to each well. Then agitate the plate on a microplate shaker and incubate at room temperature for five minutes.
Place the plate on a magnetic stand for two minutes or until the supernatant is clear. Then, without moving the plate, carefully discard the supernatant and add 200 microliters of 80%ethanol. With the plate still on the magnetic stand, incubate for 30 seconds before discarding the supernatant.
Repeat the washing process and allow the beads to dry for 15 minutes. Remove the plate from the magnetic stand and add 52.5 microliters of resuspension buffer to the beads. Using a microplate shaker, agitate the plate for two minutes at 1, 800 RPM.
Then after the shaker has stopped, incubate the plate at room temperature for two minutes. After the incubation period has ended, place the plate on a magnetic stand until the supernatant is clear. Finally, use a multi-channel pipette to transfer 50 microliters of the clear supernatant from the cleanup tray to a fresh hard-shell plate.
First follow library normalization reagents and transfer 20 microliters of the clear supernatant from the hard-shell plate to a new deep-well plate. Add 45 microliters of a magnetic bead suspension to each well of the deep-well plate and use a plate sealer to seal the plate. Next, agitate the plate on a microplate shaker at 1, 800 RPM for 30 minutes.
Place the plate on a magnetic stand for two minutes until the supernatant has cleared. Use a pipette to remove the supernatant and dispose of the supernatant into an appropriate hazardous waste container. Then remove the plate from the magnetic stand and use 45 microliters of wash buffer to wash the beads.
Agitate the plate on a microplate shaker and place the plate on a magnetic stand for two minutes. After the supernatant clears, discard the supernatant and remove the plate from the magnetic stand. Repeat the washing process with wash buffer once more.
Before removing the plate from the magnetic stand, add NaOH to each well. Then agitate the plate on a microplate shaker at 1, 800 RPM for five minutes before placing the plate on a magnetic stand until the liquid is clear. Add 30 microliters of elution buffer to each well of a new 96-well hard-shell thin-wall plate.
Finally, transfer 30 microliters of supernatant from the normalization plate to each of the wells containing elution buffer. Now, the final normalized library plate is ready to be sequenced in further experiments. 13 C.glabrata isolates obtained before and after antifungal therapy comprised of C.glabrata ATCC 90030 and twelve isolates from a clinical laboratory were studied.
The minimum inhibitory concentration, or MIC, was obtained for each isolate for nine antifungal agents. This table lists the MIC values for each isolate antifungal test condition. The presence of SNPs in genes conferring resistance in isolate correlated with elevated in-vitro MIC against antifungal drugs.
This table summarizes the SNP positions in the clinical C.glabrata isolates and the drugs that the genes confer resistance to. After watching this video, viewers can replicate this technique in a diagnostic laboratory setting for accreditation in whole genome sequencing. It highlights the important steps in DNA library preparation and the essential quality control points for accurate and reproducible results.
