Structural variants such as insertions, deletions, duplications, and inversions have in the past been more difficult to follow experimentally, and their frequencies cannot be accurately measured by amplicon sequencing. Here we provide a simple cost effective technique, which couples triplicate primer design and parallel capillary electropheresis to follow structural variant allele frequencies over time. This method was designed to complement endpoint sequencing in experimental evolution and to retrace the frequencies of emerging de novo alleles.
We hope this method will also benefit those working with intra hosts pathogen data. Demonstrating the procedure will be Jeanne Hamet, a research assistant from our laboratory. The different steps of the methods will be shown in a case where the derived allele results from an IS10 insertion in a bacterial gene called mutS.
First design a pair of primers to amplify a short amplicon on the wild type allele around the mutant insertion site. Design a third primer forward primer two within the insertion sequence to produce a slight size variable from the wild type amplicon. Begin by extracting DNA from 24-hour cultures of fixed wild type and mutant allele clones.
After quantifying the DNA, dilute each DNA extract to five nanograms per microliter with water. Fix the two samples in a 50/50 ratio. Set up a 20-microliter reaction containing 10 nanograms of DNA sample, primers, and PCR master mix.
Next, separate the PCR product by electrophoresis, using a 2%agarose gel. The size difference between the amplicons from the wild type and the mutant has to be verified on the gel. To obtain a calibration curve, begin by mixing the wild type DNA and mutant DNA in various ratios.
Dilute the PCR products to 0.1 nanograms per microliter concentration. To prepare the separation gel, mix the fresh gel and dye. Replace the inlet buffer.
Place the rinse buffer in the correct drawer location of the parallel capillary electrophoresis instrument. Add 22 microliters of the diluent marker to the wells of a 96 well plate. Now add two microliters of the diluted PCR products to each sample well.
To one Well, add a DNA size ladder from a high sensitivity kit, ranging from one to 6, 000 base pairs in size. Place the microplate in the parallel capillary electrophoresis instrument in select run on the instrument software. Analyze the results using the data analysis software.
First, identify the peaks based on their known size. Quantify each amplicon to produce a calibration curve. Finally, verify that the relative quantities of the two alleles are correctly measured.
This calibration curve is then used to calculate the quantity of structural variants after PCR amplification and parallel capillary electrophoresis. These representative results follow a disruptive insertion in the mutS gene. Knockdowns and mutS lead to a hyper mutator phenotype, allowing bacteria to sample more mutations than their base mutation rate.
Using the method presented, the mutS structural variant frequency was tracked across 1000 generations. A non-monotonic trajectory of the emerging mutant allele was revealed. The mutant allele was first detected at generation 680.
The allele then rapidly increased in frequency, reaching 67%by generation 713. This increase then stagnated, increasing only 10%over the next 53 generations, to 76%Unexpectedly, the mutant allele frequency then decreased from 76%to 49%over the course of 13 generations, after which it increased to fixation. This method will benefit those working on experimental evolution.
This protocol allows the user to take frozen archives and to explore evolutionary trajectories that are otherwise not observable with endpoint sequencing data.
