TITLE: Protocol for the detection of rare alleles in bulk seed samples
AUTHORS: Anthony Brusa, Eric Patterson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was developed for the detection of </span><span style = "font-style:italic;">Amaranthus palmeri</span><span>seeds in mixed pools with other </span><span style = "font-style:italic;">Amaranthus sp.</span><span> seeds. This is a bulk testing protocol; each pool of seeds generates </span><span style = "font-weight:bold;">one</span><span> data point, the presence or absence of </span><span style = "font-style:italic;">A. palmeri</span><span> in that sample. Testing pools should be assembled based on the relevant information (e.g. location, population, seed lot, seed supplier, etc.). Controls are extremely important for KASP based assays; this protocol requires positive, negative, and no-template controls. We strongly recommend running </span><span style = "font-weight:bold;">8 controls</span><span> for the rare allele being detected (e.g. </span><span style = "font-style:italic;">A. palmeri</span><span>) to aid in data analysis.</span></div><div class = "text-block">The protocols may be extended to any species for which appropriate species-specific KASP markers have been developed. DNA extraction protocols were performed using the MPBio FastDNA Plant Kit, but other methods may be substituted.</div></div>

[STEPS]
?. [Tissue Disruption]
Tissue disruption
?. [Tissue Disruption]
Assemble samples to be tested, partition into pools of an appropriate number of seeds (200 seeds for Amaranthus sp. testing).
?. [Tissue Disruption]
Place seeds and grinding bead into a tube suitable for tissue disruption. Avoid conical bottom tubes as this will lead to incomplete tissue disruption.
?. [Tissue Disruption]
Add 1mL lysis buffer (800 μL CLS-VF and 200 μL PPS in MPBio kit) and ensure that all seeds are submerged in buffer.
?. [Tissue Disruption]
Incubate overnight to soften seed coat for easier disruption.
?. [Tissue Disruption]
Disrupt seeds using Qiagen Tissuelyser II (or similar equipment). Recommend grinding at 30hz in 90 second intervals. Visually inspect tube after each interval to determine if further grinding is needed. Ensure that all seeds have been properly disrupted. Depending on seed size this may take upwards of 6 minutes total grinding time.
?. [DNA extraction]
DNA extractionPerform the remainder of the DNA extraction using instructions from the kit/protocol of your choice.
?. [PCR]
PCR
?. [PCR]
Create 10nmol solutions of your KASP primers. (There will be three total. Two forward primers and one reverse)
?. [PCR]
Create   of primer mix according to the following: of   forward primer (not A. palmeri) of  forward primer (A. palmeri specific) of  reverse primer of DI water
150 µl
3 µl
20 µl
45 µl
82 µl
?. [PCR]
Create your final reaction solution by combining  of KASP 2x Mastermix with  of primer mix.
432 µl
12 µl
?. [PCR]
Plate  of reaction solution into a standard 96-well PCR plate.
4 µl
?. [PCR]
Add  of DNA (approximately 10-20 ng/μL ) solution to each well. Include positive controls, negative controls and No Template Controls (NTCs). We recommend running 8 negative controls to assist in the data interpretation step below.
4 µl
?. [PCR]
Run on a Lightcycler or RT-PCR machine with the following program:15 minutes @ Touchdown program for 9 cycles at:20 sec @ 60 sec @  to  (increment with each cycle)Followed by 30 cycles of:20 sec @ 60 sec @ 30 sec @ Take an endpoint fluorescence read at 465-510 and 533-580nm for 30 seconds @
94 °C
94 °C
61 °C
55 °C
94 °C
51 °C
28 °C
28 °C
?. [PCR]
Note: 465-510 and 533-580nm correspond to the optimal fluorescence peaks of the FAM and HEX dyes respectively. At the time of writing KASP mastermix is only available for these two dyes. If your mastermix uses different dyes change the fluorescence readings as appropriate.
?. [Data interpretation]
Data interpretationNote: Standard software on lightcyclers is not designed for rare allele calling. Use the following steps instead.
?. [Data interpretation]
Export raw 465-510nm and 533-580nm reads from lightcycler software as a .csv file. Import into your statistical software of choice (R, SPSS, SAS, Mathematica, etc.)
?. [Data interpretation]
Perform an arctan transform on the ratio of 465-510nm to 533-580nm reads.  This converts your Cartesian coordinates into datapoint trajectories (angle of the datapoint relative to the origin).
?. [Data interpretation]
Generate a box-and-whisker plot using the appropriate steps in your software.
?. [Data interpretation]
A statistical comparison between the negative control and test sample will determine if a given sample contains at least one A. palmeri seed.