Feb 29, 2024
Candida auris sequencing by Illumina miSeq using Illumina DNA Prep
- 1Florida Department of Health
Protocol Citation: Brenna M McGruder Rawson 2024. Candida auris sequencing by Illumina miSeq using Illumina DNA Prep . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxjkylpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 29, 2024
Last Modified: February 29, 2024
Protocol Integer ID: 95981
Keywords: Candid auris, sequence, Illumina, miSeq, C. auris
Disclaimer
This should be considered a draft SOP and any methods or recommendations included should be validated and verified at your individual institution. The assay is not intended to diagnose or treat any medical condition. This assay is not a current laboratory developed test for any clinical testing purpose. This SOP is not endorsed by the Florida Department of Health.
Abstract
Candida auris sequencing is becoming more important for public health and surveillance. This protocol is designed to guide individuals experienced in sequencing in setting up a SOP for sequencing C. auris. This protocol is designed for Illumina short-read sequencing. While the miSeq is the described instrument it is generally not difficult to scale up or down to other instruments. We have not validated using 150x150 read lengths. The DNA prep kit is the recommended kit. The NexteraXT kit produces varying fragment sizes across samples and usually results in lower coverage and sequencing quality than samples prepared with DNA Prep.
This does not include bioinformatic analysis- however you can contact the author for more information.
Guidelines
Use no more than 100ng for DNA Prep input- The Illumina DNA Prep protocol is compatible with DNA inputs of 1–500 ng or higher. For human DNA samples and other large complex genomes, the recommended minimum
DNA input is 100–500 ng. For small genomes (eg microbial), the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling conditions accordingly).
Materials
Extracted Candida auris DNA
Illumina DNA Prep kit
Illumina miSeq
Illumina miSeq v3 reagents, 300x300
Micropipettes and micropipette tips with barrier
Safety warnings
This should be considered a draft SOP and any methods or recommendations included should be validated and verified at your individual institution. The assay is not intended to diagnose or treat any medical condition. This assay is not a current laboratory developed test for any clinical testing purpose. This SOP is not endorsed by the Florida Department of Health.
Before start
Use no more than 100ng for DNA Prep input. Using additional DNA will cause incomplete tagmentation and will result in a diverse population of fragments. The diverse population of library fragment sizes will make it difficult to accurately load the sequencing instrument and will result in poor sequencing reads.
Sample and Library preparation
Sample and Library preparation
Quantitate samples
For each sample determine the volume of sample to add that will be equal to or less than 100ng of DNA (volume must be less than 30 uL)
Add molecular grade water to a total volume of 30 uL
Vortex BLT and prepare Tagmentation MasterMix- 10uL of BLT + 10 uL TB1 for each sample plus overage
Add 20 uL Tagmentation MasterMix to DNA and mix well by pipetting or plate shaker
On a thermocycler run the Tagmentation step: 55°C for 15 minutes
Check TSB for precipitates, warm if needed. Add 10uL of TSB to each samples.
On a thermocycler run the Post-Tagmentation step: 37°C for 15 minutes.
Place on a magnet for 3 minutes and discard the supernatant
Remove the plate from the magnet and add 100 uL of TWB and mix gently
Place on the magnet for 3 minutes and discard the supernatant
preform one more wash (steps 10-11)
Remove from the magnet and add 100uL of TWB and mix gently. Place on magnet for at least 3 minutes
Prepare the PCR Mastermix- 20uL EPM + 20 uL water for each sample plus overage
Once the PCR Mastermix is prepared remove the final TWB, making sure to remove as much excess TWB as possible
Add 40 uL of the PCR Mastermix and re-suspend the pellet by pipetting or using the plate shaker
Add 10uL of Index and mix well
On a thermocycler run the suggested PCR for 5-10 cycles.
Note: for high amounts of input DNA fewer PCR cycles are recommended.
68°C for 3 min
98°C for 3 min
5-8 cycles for 100ng
98°C for 45 sec
62°C for 30 sec
68°C for 2 min
68°C for 1 min
Hold at 4°C
Post-Library Clean up
Post-Library Clean up
Clean up can be done either via the small fragment cleanup protocol or the dual size selection.
Pool preparation
Pool preparation
Samples can be quantitated and pooled in equal amount or can be pooled by equal volume.
Pools should be quantitated and the average fragment size should be assessed to effectively load the instrument.
Loading
Loading
Recommended loading concentration for a miSeq v3 is 10-12 pM. The goal should be 1000-1200 k/mm2.