Applied BiosystemsTM genetic analysis systems and consumables have long been recognized as reliable and widely used tools for Sanger sequencing. Among their product line, the BigDye Terminator v3.1 Cycle Sequencing Kit has established itself as a trusted leader for over 25 years.
This kit is specifically designed to address the demands of various applications, including challenging templates. In this blog, we will explore a comparative study that evaluates the performance of the BigDye Terminator v3.1 Kit against an alternative product for sequencing challenging templates, such as high GC rich content and homopolymer regions.
Figure 1. Raw data are shown for reactions made with 0.5μL of each Cycle Sequencing kit for both forward and reverse sequencing reads of a rich GC rich template. (A,B) BigDye kit has high 5’ to 3’ signal throughput the sequences. (C,D) Shows a steady to drastic decline in signal intensity toward the 3’ end of the reads using alternative Product M kit.
Sequencing High GC Rich Content
Sequencing DNA templates with high GC content can present significant challenges.
To assess the performance of the BigDye Terminator v3.1 Kit and the alternative product (referred to as Product M), a study was conducted. The results showed that the BigDye Terminator v3.1 Kit produced higher signal intensities for both forward and reverse reads, as demonstrated in Figures 1A and 1B.
In contrast, Product M exhibited a drastic decline in signal intensity, as depicted in Figures 1C and 1D. Overall, the BigDye Terminator v3.1 Kit outperformed Product M by providing higher signal intensity and longer contiguous read lengths (CRL) when sequencing templates with high GC content.
Sequencing Homopolymer Regions
Homopolymer regions, characterized by repetitive stretches of the same nucleotide, can be particularly challenging for accurate sequencing.
The enzymes used in the polymerase reactions can lead to polymerase slippage, resulting in sequencing errors known as N-1 errors. To evaluate the performance of both kits in sequencing homopolymer regions, a string of thymine (T) was used as the template.
The results, as shown in Figure 2, demonstrated that the BigDye Terminator v3.1 chemistry exhibited less polymerase slippage compared to Product M. This characteristic enabled the BigDye Terminator v3.1 Kit to sequence through homopolymer regions more effectively, producing higher-quality data that can be analyzed using secondary analysis software. In contrast, Product M showed a loss of base calling after the homopolymer repeat, leading to poor quality data that cannot be adequately analyzed.
Figure 2. Raw signal plot are shown for reactions made with 0.5μL of each Cycle Sequencing kit for forward sequencing reads of a thymine (T) homopolymer repeat region. (A) BigDye Terminator v3.1 chemistry shows base calling after homopolymer repeat with a more robust polymerase performance whereas (B) Product M chemistry results in loss of base calling after the homopolymer repeat due to polymerase slippage.
Summary
In summary, the comparative study revealed the superiority of the BigDye Terminator v3.1 Cycle Sequencing Kit over the alternative product for sequencing challenging templates.
It consistently provided longer contiguous read lengths (CRL) and higher signal intensities across a wide range of template types, including those with high GC content. Additionally, it demonstrated improved accuracy in handling homopolymer stretches, resulting in more reliable and usable data for further analysis.
The BigDye Terminator v3.1 Cycle Sequencing Kit is a reliable tool that enables accurate results across a broad range of sample types.
Conclusion
The BigDye Terminator v3.1 Cycle Sequencing Kit continues to be a trusted choice for Sanger sequencing.
Its outstanding performance in challenging sequencing scenarios, such as high GC content and homopolymer regions, makes it a preferred option for researchers seeking reliable and high-quality results. With its track record of over 25 years and more than 15,000 publication references, the BigDye Terminator v3.1 Kit reinforces its position as a robust and dependable tool in the field of genetic analysis.
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