BEST: Barcode Enabled Sequencing of Tetrads

The overall goal of this procedure is to create a large number of recombinant progeny from a yeast cross in a manner that retains the information about which progeny were derived from the same original tetrad. This is accomplished by first transforming the heterozygous diploid strain to be used in the cross with a plasmid library that contains a tetra specific barcode and a sporulation specific fluorescent reporter. The second step is to transfer the barcoded cells to sporulation medium.

This allows tetras to form and induces the expression of the GFP fluorescent reporter in Tetras, but not in cells that fail to sporulate. Next, a fax sorter separates the fluorescently labeled tetras away from other cells and deposits them directly into an agar plate where the outer coating of the tetras is enzymatically digested. The final step is to use glass beads to separate and spread the spores of the digested tetras across the surface of the agar plate, allowing each spore to form a single colony.

Ultimately, the strains obtained using this method will be genotyped using a high throughput multiplexed DNA sequencing protocol that will read the tetra specific barcode of each strain. The barcodes are then used to identify each of the four strains that resulted from a single miotic event. The main advantage of this technique over existing methods like manual Ted dry dissection using a micro manipulator, is that one researcher can isolate several thousand recombinant progeny in just a few hours.

The diploid yeast cells used in this study have been previously transformed with a library of barcoded plasmids as described in the accompanying manuscript and stored at negative 80 degrees Celsius to revive cells from the frozen stalks. Grow them overnight in five milliliters of YPD plus 200 micrograms per milliliter G four 18 at 30 degrees Celsius with agitation on the following day. Wash the cells from the overnight cultures three times in PBS and then resuspend cells to a final volume of two milliliters in sporulation medium, plus 200 micrograms per milliliter.

G four 18 Agitate sporulation cultures at room temperature. Monitor sporulation progress daily until new tetrads have stopped forming. Typically, this takes between two to four days once the cultures contain well-formed tetrads and relatively few dyads.

Remove the cultures from agitation and leave at room temperature for at least seven days before tetrad sorting. The most difficult aspect of this procedure is getting good separation of spores. We found empirically that allowing the sporulate cultures to sit at room temperature without agitation for at least a week prior to tetrad sorting greatly improve spore separation.

The tetrads from the sporulation culture can be detected and isolated by fluorescence activated cell sorting or fax because the barcoded plasmids in the diploid cells also carry a sporulation specific GFP fusion protein. The protocol described here was developed for a fax aria to two. To begin this procedure, load the sporulated culture onto the fax sorter and set up two forward scatter and side scatter gates to exclude debris, clumped cells and multiple cells per droplet.

There should be a population of fluorescent cells, which when using the PS two GFP reporter construct includes dyads and tetrads gait, the GFP versus FSC to include these fluorescent events. Depending on the strain background, it may be necessary to include an additional gait to remove clumps that include tetras and other cells. Inspect an FSC histogram of the fluorescent events.

In this example, events in the left peak are primarily tetras, and events in the right peak are generally tetras with an attached bud apply a final gate that includes events with lower FSC that are mostly tetras indicated by the red bars. Check the efficiency of the gating regime by sorting approximately 1, 500 tetras onto a microscope slide and counting the proportion of tetrads to non tetrads. Using a phase contrast microscope at 400 times magnification, the percent of tetrads can be accurately estimated by counting only approximately 200 sorted events, but sorting approximately 1, 500 reduces the amount of time spent searching for cells on the microscope slide.

These bright field images add 600 times magnification. Show examples of tetrads on the left and non tetrads on the right after facts sorting. Periodically check that the sorter is accurately reporting the number of events by sorting 25 tetras onto a microscope slide and examining the cells contained in the droplet using phase contrast microscope at 400 times magnification.

There should be 25 tetrads in the droplet. Repeat three or four times to assess the variance of the fax instrument in that run. Prepare the agar plates for this procedure by warming the desired number of YPD plus G four 18 plates for at least 1.5 hours in a 37 degree Celsius incubator positioned near the fax sorter.

A key feature of the sorting protocol is the ability to accurately target fax sorted tetrads to a specific location on an agar plate that contains a pool of X solution. To do this first place a landmark 96 well plate on the automated cell deposition unit of the sorter. Next, prepare a sort layout that will sort 25 tetras into one well of a 96.

Well format sorting 25 tetras per plate yields well separated colonies. Choose a well near the middle of the plate. For example, well D five.

Bring a stack of 10 agar plates from the 37 degree Celsius incubator to the sorter. Take one agar plate and pipette 25 microliters of zy solution onto the agar near the center of the plate. Then place the plate on the landmark 96 well plate, aligning the X droplet over the target.

Well that was chosen earlier. Well D five. Start the sort and deposit 25 tetras into the middle of the zy droplet on the agar plate.

Remove and cover the plate. Repeat the sorting for each plate until 25 tetrads have been sorted into zamo droplets on each plate in the stack. Return the stack of tetra containing plates to the 37 degree Celsius incubator.

Bring the next stack of 10 plates to the sorter and repeat the sorting procedure. Spores can be separated after the tetrad containing agar plates have been incubated at 37 degrees Celsius for 1.5 hours. To begin this procedure, add 15 to 25 sterile three millimeter glass beads to each plate, and shake the plates as two stacks of five plates for three to five minutes.

The best spore separation is achieved by moving the plates rigidly from side to side or front to back. Do not move the plates such that the beads swirl around the outside edge of the plate. Leave the beads on the plates when finished.

Place the stack of plates with beads face up in a 30 degree Celsius incubator. Retrieve the next stack of plates from the 37 degrees Celsius incubator and repeat the spore separation procedure using the beads. Incubate all plates overnight at 30 degrees Celsius on the day after separating the spores.

Carefully remove the plates from the 30 degrees Celsius incubator without disturbing the glass beads. Small colonies should have formed on the plates. Remove the glass beads by carefully and quickly inverting the plates over a deep container.

Tapping the bottom of the plate while it is inverted, can help dislodge beads that are stuck to the plate. The containers should be deep enough to catch the beads without allowing them to bounce up and hit the agar plate. Count the number of colonies on each plate and use the number of colonies to estimate the efficiency of spore separation and recovery.

For example, 25 tetrads per plate for a cross with near 100%viability could yield a total of 100 colonies per plate from plates with sufficient numbers of colonies such as greater than 65 for a high viability. Cross transfer cells from each colony into a separate well of a 96 well plate containing YPD liquid plus G four 18. Also make note of which wells contain colonies from the same agar plate.

This information will be used later to aid the tetra assignment. Software best can greatly improve the speed with which spores from tetrads can be isolated. Although the current iteration of the method has some loss of efficiency compared to manual dissection, the binomial distribution was used to calculate the expected fraction of tetrads with at least one successfully recovered spore producing 1, 2, 3, or four colonies based on the proportion of all spores successfully recovered.

This graph can serve as a rough guide to the number of strains that will ultimately be assembled into three or four spore tetras as observed here. The number of complete tetras recovered increases as the number of colonies per plate increases in this study. 25 rads from across with spore viability close to 100%as determined manual dissection were plated on 149 agar plates.

The maximum number of colonies observed per plate was 80 indicating that 20 spores failed to form discrete colonies. The mean number of colonies per plate was 62 4, 354 colonies from 61 plates containing 65 colonies or more were harvested and sequenced. Of the 3, 700 strains that passed sequencing quality control, 2, 665 or 72%could be computationally assigned into three or four spore tetrads Once mastered.

This technique can be used to process more than a thousand tetrads per hour. Although for precisions longer than three hours, you'll need assistance.