Isolation of single-cells while retaining whole genome integrity is challenging, whether using fluorescence-activated cell sorting, microfluidics, laser-capture microdissection, micromanipulation, dilution, or other techniques.
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Isolation of single-cells while retaining whole genome integrity is challenging, whether using fluorescence-activated cell sorting, microfluidics, laser-capture microdissection, micromanipulation, dilution, or other techniques. And whole genome amplification (WGA) can introduce amplification bias and artefacts due to the use of random primers in excess and/or PCR. Consequently single-cell WGA can fail or give poor genome coverage, and it has become common to compensate by preparing and sequencing many replicates. Considering the cost and time required for next generation sequencing (NGS), it is important to check success of amplification and ideally genome coverage after WGA before sending samples to sequencing. Several methods can be used to do so, with varying degree of information, input requirement, speed and cost.
The quickest and simplest method for checking amplification success is to quantify the obtained DNA. This can be done with directly with 260nm UV/Vis absorbance, by fluorescence with PicoGreen, or by any other DNA quantification methods. In the case of 260nm UV/Vis absorbance, it is critical to purify the obtained DNA to avoid oligonucleotide contamination. Running an agarose gel is another similar method. All of these methods are fast, cost-effective, and low-input but they only give information about the quantity of DNA obtained but not about DNA quality and genome coverage. It is useful to quantitate DNA for all the other methods and for library preparation in any case.
Specific profiling arrays can be used such to attempt to assess coverage in known samples, such as CGH or SNP arrays. These will check specific copy number variations or polymorphisms for a chromosome, and therefore should identify if there are no copies of target chromosomes in the sample. However, if a DNA sample is novel, mutated, or relatively unknown it’s difficult to employ these methods due to their specificity. Additionally, only chromosomes being assessed for CGH or SNP can be detected.
A widely used method to check the quality and coverage of obtained DNA is to do low depth sequencing, typically before doing high depth sequencing. This approach gives the most comprehensive information about DNA quality and genome coverage, and can assess potential DNA contamination with the right analysis tool. The drawback is that it is time-consuming and can be fairly expensive. It also requires enough obtained DNA to prepare two libraries (one for low depth sequencing QC and one for deep sequencing), with the risk of preparing libraries for sample with poor genome coverage.
Another method consists of using homemade specific primer-pairs to check for the presence of small regions on some chromosomes by PCR. The results can be visualized quickly on an agarose gel, with a Bioanalyser or by quantitative PCR. Finding and validating such primer-pairs is not straightforward, but once optimised this methods is a fast, cost-effective, and low-input way to QC single-cell WGA. Depending on the number of primer-pairs, the information about genome coverage can be quite limited however. And most protocols rely on a couple of primer-pairs only. For developing CovCheck QC kit, 24 primer-pairs were validated to cover all 22 autosomes and sexual chromosomes X and Y. Detection of most (or very little) of the 24 PCR amplicons tightly correlate with good (or poor) genome coverage from NGS, making it a balanced methods for information, input, speed and cost.
As we develop WGA kits for single-cells and other source of rare DNA we do a lot of amplifications and a lot of sequencing. To save time and minimize unnecessary costs, amplification QC is critical for us, and we hope our expertise can help you choose the best QC method for each of your projects or workflows.