Hello,
Since a large part of my budget will go on NGS and I don't have the money to repeat it, I want to make sure that it will work.
The PCR products look good on the gel (nice bands of the expected length), but I was also wondering whether it's feasible to send a few samples for Sanger Sequencing to verify the product, before I spend all my money of an Illumina run.
Thanks :)
Ryan Gourlie
We will often confirm the species identity prior to NGS via PCR product sequencing and BLAST searches. I think it's fairly common to and good practice to know what you're sequencing before hand.
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Kais Khudhair al Hadrawi
Accuracy Check: Sanger sequencing provides a highly accurate and reliable method for verifying the sequence of a PCR product. It can confirm the presence of specific target sequences, identify mutations, and detect any errors or artifacts introduced during PCR amplification. Confirmation of Amplicon Identity: Sanger sequencing can confirm that the PCR product corresponds to the expected target sequence. It ensures that the correct region of interest has been amplified before proceeding with downstream NGS analysis. Quality Control: Sanger sequencing serves as a quality control step to validate the integrity of the PCR product and the accuracy of the amplification process. It helps identify any potential issues such as contamination, primer dimers, or non-specific amplification that may affect the reliability of NGS results. Cost-Efficiency: Sanger sequencing is generally more cost-effective than NGS for verifying individual PCR products or confirming a small number of samples. It provides a quick and reliable way to validate PCR results before investing resources in NGS library preparation and sequencing. Complementary Approach: Combining Sanger sequencing with NGS allows researchers to leverage the strengths of both technologies. Sanger sequencing provides high accuracy for verifying individual amplicons, while NGS offers high-throughput sequencing of multiple samples simultaneously..
Verifying PCR results with Sanger sequencing prior to Next-Generation Sequencing (NGS) is a common practice in many research and clinical laboratories. Here's why:
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