The overall goal of this procedure is to successfully use the Infinium assay in order to process a genotyping bead chip. This is accomplished by first amplifying, fragmenting and isolating the DNA. The second step is to hybridize the denatured DNA strand to oligonucleotides attached to beads on the array.
Next, the oligonucleotide attached to the bead is stained and selectively extended by one base. The final step is to scan the bead chips to detect the labeled nucleotide. Ultimately, the genotype results are used to determine the alleles present at the site of interest.
Visual demonstration of this method is critical because many of the steps for staining and loading the bee chips are difficult to learn. Many of the maneuvers are not found in other genotyping methods and many of the equipment share very similar sounding names. One day prior to amplification prepare the DNA dispense 200 nanograms of DNA per well into a deep well 96 sample plate.
At least 96 samples must be plated to ensure no reagent will be wasted. Label the plate with a barcode sticker supplied by the kit and centrifuge it in order to normalize the volumes. Leave the samples in a drawer or fume hood overnight to evaporate the liquid.
Cover the plate loosely with a lid or paper towel to keep out dust on the following morning. Check that the wells are dry user reagent basin and a 10 microliter eight channel pipette to dispense four microliters of DNA Resus buffer into each well to rehydrate the samples. If care is taken not to touch the liquid, it is not necessary to discard pipette tips between each column.
Next, dispense 20 microliters of MA one reagent into each well of the plate using an eight channel pipette and a fresh reagent basin. Cover the plate with a reusable seal, pulse centrifuge and vortex for one minute at 1600 RPM on a microplate shaker incubate at room temperature for at least 30 minutes. The remaining steps for amplification will not be shown in this video, but the protocol can be found in the accompanying manuscript.
After all reagents have been added, place the plate in a properly calibrated oven set to 37 degrees Celsius for 20 to 24 hours. Following amplification, the DNA samples are fragmented and precipitated as described in the manuscript following precipitation in a high speed centrifuge. Inspect the bottom of the plate without inverting it.
To confirm the samples are precipitated in a blue pellet, discard the lid and quickly remove the liquid by inverting the plate and tapping it forcefully on the bench top covered in paper towels. Once the plate is inverted, take care not to revert it. While any liquid remains repeatedly, tap the plate against the bench top until all liquid is removed.
Set the plate on a test tube rack inverted to dry incubate at room temperature for one hour. Subsequently, the DNA is resuspended to begin the hybridization procedure. Place a 96 well plate containing resuspended DNA on a heat block at 95 degrees Celsius for 20 minutes.
To denature the DNA after 20 minutes, remove the sample plate from the heat block and incubate at room temperature for 30 minutes while the sample plate is cooling, prepare the hive chamber. One chamber will be needed for every four bee chips processed. Place a rubber mat on the top of the hive chamber base aligning the thicker hole with the front of the base.
The barcode symbol etched into the base should still be visible. Using a 1000 microliter pipette dispense 400 microliters of PB two reagent into each of the eight humidifying reservoirs carved into the base. Place the hi chamber lid on the base and clasp both ends by placing two clasps on diagonally opposite sides.
First place four Hi chamber inserts on the bench for each hi chamber used just before the completion of the 30 minute. Cool down. Open the silver bead chip packs.
Remove the chips from their clear plastic sleeves without touching the beads. Place each bee chip on a hi chamber insert. Orienting the chip barcode with the barcode symbol etched into the insert's top surface.
The next step, which is the loading of the bee chips, may be the most critical step of the protocol. Doing it correctly requires visualization of the sample transfer from DNA plate to bee chip ahead of time, Peel off and discard the lid of the sample plate. A multichannel pipette may be used to dispense liquid on the chips, but forethought must be taken to aspirate only the number of samples that can be safely placed on the bee chip as the number of rows on a plate does not always match the number of rows on a chip.
Take 15 microliters of sample from the sample plate and slowly dispense on the inlet to the array. Extraordinary care must be taken to place the correct sample on the correct bee chip in the correct position matching the bead chip. Order recorded earlier.
Open the hi chamber and place the inserts over the humidifying reservoirs. The barcode of the chip should be placed over the barcode etch into the hive chamber base. Replace the hive chamber cover and close all four clasps by closing the clasps on diagonally opposite sides.
First, incubate the hi chamber in a 48 degrees Celsius oven for 16 to 24 hours at the completion of hybridization, the hi chambers are removed from the oven and incubated at room temperature for 25 minutes to cool down. When the hi chambers have cooled, open one hi chamber. Remove the cover seal from a bee chip by grasping the seal at one corner and gently peeling diagonally.
Once the seal is removed immediately place the chip on a bee chip tray and submerge in PB one without touching the beads. Repeat until the tray is filled with bead chips. Taking care not to let any chip dry.
Gently agitate the tray to remove any bubbles and leave the chips submerged for one minute. Agitate the tray again. Move the tray of bee chips into a second wash dish filled with PB.One gently agitate and let it soap for one minute.
Then agitate again in the liquid flow through assembly station. Place four black plastic flow through braces in the grooves. Fill the station with PB one until the liquid nearly reaches the height of the eight assembly brackets one at a time.
Remove bead chips from the tray and place them into the assembly station on a plastic flow through brace, the chip barcode should be aligned above the barcode symbol etched into the assembly station. Once four chips are in the assembly station, place a clear plastic spacer on top of each bead chip. The outer edges of the spacer should surround the brackets within the assembly station.
Place the plastic assembly bar in the assembly station by fitting it over the grooves. Gently place a glass slide on top of the plastic spacer by pushing the rear end of the slide against the assembly bar and slowly lowering the front into the liquid. The groove on the top of the slide should face downwards directly above the barcode of the chip, leaving a gap between the bee chip and the slide at the barcode end.
After checking that there are no bubbles between the chip and slide snap two metal clasps around the glass. Slide one towards the barcode end and one towards the rear. The edges of the plastic clasps should grip the plastic flow through brace under the chip.
This figure shows a complete flow through assembly diagram. Once all beads are in their flow through assemblies, take a pair of surgical scissors and cut both ends of the spacer off as near to the glass slide as possible Once the chamber rack is at 44 degrees, begin this procedure by placing a bee chip flow through assembly on the chamber rack by sliding the assembly down and hooking the brace at the top. The backside of the bee chip should be touching the chamber rack.
The glass slide should be facing out with the groove at the top to form a reagent reservoir. Once all bee chip assemblies have been placed on the chamber rack, use a 200 microliter pipette to add 150 microliters of RA one reagent to the flow through assemblies. By dispensing the liquid into the glass reservoir, the reagent will naturally flow down the slide and exit at the base of the assembly, allowing future reagents to be placed directly into the reservoir.
The remaining steps for staining an extension will not be shown. Please refer to the protocol in the accompanying manuscript at the completion of the staining and extension. Disassemble the flow through assembly by inserting a thin metal bar between the clasps and the brace.
Then pivoting. Set aside the glass slide and remove the bee chip immediately placed a bee chip in a vertical bee chip tray and submerge in PB one, repeat for every flow through assembly. Taking care to face each bee chip in the same direction and minimizing their exposure to air.
Subsequently complete this procedure. According to the protocol in the accompanying text, a properly processed bee chip should display bright and distinct red and green laser light intensities when scanned because the labeled nucleotides that were attached during the extension and staining steps will fluoresce under the lights of the two lasers. Shown here is a standard genomic DNA sample successfully hybridized to a custom panel SNP genotyping array sections passing intensity.
QC will highlight green on the bee chip display to the left sections. Failing intensity. QC will highlight red on the bee chip display.
Once scanning is complete, the will overlay the red and green displays. A zoomed in image is shown here as these nucleotides selectively extend the beads oligo nucleotide chain hybridized to the fragmented DNA strand. And as the nucleotide chains are designed to terminate at the site of the variant, the color and intensity of the resulting signal can be used to determine the alleles present at the SNP site.
The scanner output files are imported into the Genome Studio Analysis software, which generates cluster profiles. This is an example of a valid SNP with three distinct clusters representing aa, A, B and B, B genotypes colored red, purple, and blue respectively. This intensity plot shows the SNP that required editing the black middle cluster, which should be homozygous.
AB is left uncalled. The BB cluster is mistakenly called ab. The last intensity plot shows a poor performing SNP.
No genotypes can be obtained from this intensity plot as no distinct clusters exist. While attempting this procedure, it is important to remember to process the bee chips quickly. Do not expose them to air for any extended period of time.
