An inability to read through long strings of C or G and the ability of the template to form loops when the polymerase may read across the neck of the loop to form an apparent deletion can be a problem. To some extent if you are running your own sequencing reactions then sequencing at a high annealing temperature and high extension temperature can help minimise secondary structure and get better results. Also sequencing in a buffer with dmso and betaine will minimise secondary structure. Commercial sequencing companies will take care to use the most robust sequencing kit and conditions if warned about the high GC. Sequencing in the presence of base analogues will also help. sequencing with nanopore technology has fewer problems with high Gc as does pyrosequencing. If the GC content is very high then the electropherogram will show a weakening signal as the sequencing kit runs out of C and G dyedeoxy bases which can be a problem with long sequences.

Sometimes the single stranded dna template forms a loop so stable that the the enzyme simply stops extending and leaves the latter part of the sequence unsequenced.

There can also be problems if the primers are high GC and exo sap is used as the primer removal method prior to sequencing where one primer can be resistant to the exonuclease and the result is 2 overlapping sequences

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

High GC content in a gene can indeed be problematic for sequencing due to its stable, tightly-bound structure, which can hinder the denaturation and reannealing processes essential for sequencing. Common errors include poor sequence quality, incomplete reads, and low coverage in GC-rich regions [1][3][4]. This can result in inaccurate or missing data. Troubleshooting strategies include optimizing PCR conditions with additives like DMSO or betaine, using high-fidelity polymerases such as Kapa HiFi, and employing sequencing platforms with better performance on GC-rich templates [2][6][7]. Adjusting annealing temperatures and extending the denaturation step may also improve the accuracy of sequencing GC-rich genes [5][8].

Reference

[1] Benjamini, Y., & Speed, T. (2012). Summarizing and correcting the GC content bias in high-throughput sequencing. Nucleic Acids Research, 40, e72 - e72.

[2] Chen, Y., Liu, T., Yu, C., Chiang, T., & Hwang, C. (2013). Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly. PLoS ONE, 8.

[3] Shin, S. C., Ahn, D., Kim, S., Lee, H., Oh, T., Lee, J. E., & Park, H. (2013). Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes. PLoS ONE, 8.

[4] Lee, H., Lee, B., Kim, D., Cho, Y., Kim, J., & Suh, Y. (2021). Detection of TERT Promoter Mutations Using Targeted Next-Generation Sequencing: Overcoming GC Bias through Trial and Error. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 54, 75 - 83.

[5] Lecomte, E., Saleun, S., Bolteau, M., Guy-Duché, A., Adjali, O., Blouin, V., Penaud-Budloo, M., & Ayuso, E. (2020). The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers. Biotechnology Journal, 16.

[6] Kumar, A., & Kaur, J. (2014). Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model. Molecular Biology International, 2014.

[7] Thorner, A., Sunkavalli, A., Lin, L., Jones, R., Schubert, L., Ducar, M., Adusumilli, R., Dolcen, D. N., Ziaugra, L., Lepine, J. R., Macconaill, L., Hahn, W., Meyerson, M., & Hummelen, P. (2014). Abstract 3583: Reducing GC-bias and improving coverage distribution in Illumina sequencing using the Kapa Biosystems library construction method. Cancer Research, 74, 3583-3583.

[8] Stoler, N., & Nekrutenko, A. (2021). Sequencing error profiles of Illumina sequencing instruments. NAR Genomics and Bioinformatics, 3.