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PowerPlex® ESI Systems: D12S391 and D2S441. Resolving Alleles With Single-Base-Pair...

PowerPlex® ESI Systems: D12S391 and D2S441. Resolving Alleles With Single-Base-Pair Size Differences

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Andy Hopwood1 and Ariane Davidson2

1Promega Corporation, 2Biological Industries, kibbutz Beit Haemek, Israel

Publication Date: 2013

All commercially available STR analysis systems include loci that exhibit microvariant alleles, which differ in length by just a single base pair. The resolution capability of the instrumentation decreases as the STR length increases, and where alleles with a single-base-pair size difference are in the higher-molecular-weight range (for example, over 300bp), there can be occasional issues with resolving the alleles, particularly when one of the alleles is present at a much lower proportion than the adjacent allele. This has been noted where separation of alleles is performed using Applied Biosystems® 3130 or 3500 capillary electrophoresis (CE) instrumentation, maintained in accordance with the manufacturer’s guidelines, and where data are analysed using GeneMapper® ID software, version 3.2, or GeneMapper® ID-X software, version 1.2.1. This rare phenomenon has been observed with the Promega PowerPlex® ESI Systems at the loci D12S391, D2S441 and D1S1656.

In some cases, the data analysis parameters in the GeneMapper® software can be adjusted, in accordance with the Applied Biosystems GeneMapper® user manual, to analyse the data. For example, the polynomial degree and peak window can be modified to adjust the sensitivity of peak detection (Figure 1, Panels A and B). However, we have observed that alleles separated by a single base cannot always be fully resolved using the GeneMapper® software in some samples despite the alleles being visually different on the electropherogram (Figure 2). We have found that the SoftGenetics analysis software, GeneMarker® HID, version 2.4.0, can be useful in analysing some of these samples (Figure 3).

Hopwood Figure 1Figure 1. Analysis of PowerPlex® ESI 16 System data using GeneMapper® ID software.

Panel A. Analysis of a sample using a peak window size of 7 or above results in the 12 allele being unavailable for automatic or manual labelling. Panel B. Analysis parameters modified to peak window size of 5 allows automatic labelling of the 12 allele with this sample.

Panel A. Analysis of a sample using a peak window size of 7 or above results in the 12 allele being unavailable for automatic or manual labelling. Panel B. Analysis parameters modified to peak window size of 5 allows automatic labelling of the 12 allele with this sample.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 1.jpg?la=en
Hopwood Figure 2Figure 2. A mixed DNA sample exhibiting 18, 19.3, 20 and 25 alleles at D12S391.

The 19.3 and 20 alleles are eluted close together, and the GeneMapper® ID-X software, version 1.2.1, allows selection of only the 20 allele, despite the presence of both the 19.3 and 20 alleles.

The 19.3 and 20 alleles are eluted close together, and the GeneMapper® ID-X software, version 1.2.1, allows selection of only the 20 allele, despite the presence of both the 19.3 and 20 alleles.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 2.jpg?la=en
Hopwood Figure 3Figure 3. The same data as in Figure 2 analysed using SoftGenetics GeneMarker® HID software.

The D12S391 18 and 20 alleles in this sample file were called automatically. The 25 allele is highlighted in red, signifying that it requires checking. The 19.3 allele can be called by selecting the peak and manually adding the call.

The D12S391 18 and 20 alleles in this sample file were called automatically. The 25 allele is highlighted in red, signifying that it requires checking. The 19.3 allele can be called by selecting the peak and manually adding the call.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 3.jpg?la=en

Where this issue with data analysis of alleles separated by a single base occurs, we recommend that wherever possible you:

  • check the resolution of the CE instrument, make improvements if necessary and re-run the sample.
  • reprocess the DNA samples using the PowerPlex® ESX System, where the loci prone to this issue are of a lower molecular weight and more likely to exhibit good resolution on the CE instruments (Figure 4).
  • analyse the CE data using an alternative software (e.g., GeneMarker® HID, version 2.4.0, from SoftGenetics).

Hopwood Figure 4Figure 4. The same sample as in Figure 2 amplified using the PowerPlex® ESX System showing resolution of the 19.3 and 20 alleles at D12S391 at lower molecular weight.

Note that the size difference between the two alleles is 1.43bp rather than the expected approximately 1bp. The 20 allele is likely to be comprised of a different repeat sequence compared with alleles 18 and 19.3.

Note that the size difference between the two alleles is 1.43bp rather than the expected approximately 1bp. The 20 allele is likely to be comprised of a different repeat sequence compared with alleles 18 and 19.3.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 4.jpg?la=en

Acknowledgments

The authors would like to thank the members of the Forensic Biology Laboratory, Division of Identification and Forensic Science (DIFS), at the Israel Police for their contribution of data.

How to Cite This Article

Hopwood, A. and Davidson, A. PowerPlex® ESI Systems: D12S391 and D2S441. Resolving Alleles With Single-Base-Pair Size Differences. [Internet] 2013. [cited: year, month, date]. Available from: http://ita.promega.com/resources/profiles-in-dna/2013/powerplex-esi-systems-resolving-alleles-with-single-base-pair-size-differences/

Hopwood, A. and Davidson, A. PowerPlex® ESI Systems: D12S391 and D2S441. Resolving Alleles With Single-Base-Pair Size Differences. Promega Corporation Web site. http://ita.promega.com/resources/profiles-in-dna/2013/powerplex-esi-systems-resolving-alleles-with-single-base-pair-size-differences/ Updated 2013. Accessed Month Day, Year.

Contribution of an article to Profiles in DNA does not constitute an endorsement of Promega products.

PowerPlex is a registered trademark of Promega Corporation.

Applied Biosystems and GeneMapper are registered trademarks of Applied Biosystems. GeneMarker is a registered trademark of SoftGenetics Corporation.

Figures

Hopwood Figure 1Figure 1. Analysis of PowerPlex® ESI 16 System data using GeneMapper® ID software.

Panel A. Analysis of a sample using a peak window size of 7 or above results in the 12 allele being unavailable for automatic or manual labelling. Panel B. Analysis parameters modified to peak window size of 5 allows automatic labelling of the 12 allele with this sample.

Panel A. Analysis of a sample using a peak window size of 7 or above results in the 12 allele being unavailable for automatic or manual labelling. Panel B. Analysis parameters modified to peak window size of 5 allows automatic labelling of the 12 allele with this sample.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 1.jpg?la=en
Hopwood Figure 2Figure 2. A mixed DNA sample exhibiting 18, 19.3, 20 and 25 alleles at D12S391.

The 19.3 and 20 alleles are eluted close together, and the GeneMapper® ID-X software, version 1.2.1, allows selection of only the 20 allele, despite the presence of both the 19.3 and 20 alleles.

The 19.3 and 20 alleles are eluted close together, and the GeneMapper® ID-X software, version 1.2.1, allows selection of only the 20 allele, despite the presence of both the 19.3 and 20 alleles.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 2.jpg?la=en
Hopwood Figure 3Figure 3. The same data as in Figure 2 analysed using SoftGenetics GeneMarker® HID software.

The D12S391 18 and 20 alleles in this sample file were called automatically. The 25 allele is highlighted in red, signifying that it requires checking. The 19.3 allele can be called by selecting the peak and manually adding the call.

The D12S391 18 and 20 alleles in this sample file were called automatically. The 25 allele is highlighted in red, signifying that it requires checking. The 19.3 allele can be called by selecting the peak and manually adding the call.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 3.jpg?la=en
Hopwood Figure 4Figure 4. The same sample as in Figure 2 amplified using the PowerPlex® ESX System showing resolution of the 19.3 and 20 alleles at D12S391 at lower molecular weight.

Note that the size difference between the two alleles is 1.43bp rather than the expected approximately 1bp. The 20 allele is likely to be comprised of a different repeat sequence compared with alleles 18 and 19.3.

Note that the size difference between the two alleles is 1.43bp rather than the expected approximately 1bp. The 20 allele is likely to be comprised of a different repeat sequence compared with alleles 18 and 19.3.

/~/media/images/resources/figures/profiles in dna/2013/hopwood figure 4.jpg?la=en

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