The genome, which contains the genetic information of an organism, comprises various combinations of A, T, G, C nucleotides. Depending on these sequences’ characteristics, experiments and analyses might face challenges, impacting the accuracy of sequencing and analysis. Therefore, it is crucial to consider the characteristics and aspects of these so-called ‘hard-to-capture’ regions when designing a target capture panel.
1. Inclusion of repetitive sequences
Repetitive sequences, involving the repetition of one or more nucleotide sequences multiple times, can range from a few nucleotides to thousands. Excessive repetition or the length of these regions can induce errors during PCR amplification or sequencing processes, and it can also lead to reduced alignment accuracy in NGS data analysis.
2. Presence of duplicated sequences
Duplicated sequences denote areas where different regions in the genome have identical or highly similar nucleotide sequences. Capturing targets within these regions can diminish probe efficiency due to potential binding of probes to multiple duplicate locations. Moreover, indistinguishable large duplicated sequence regions can lead to alignment failures in NGS data analysis.
3. Extremely high or low GC ratios
The GC ratio signifies the proportion of G and C nucleotides within a specific sequence. In DNA pairing, A and T exhibit relatively weaker bonding compared to G and C. Higher GC ratios intensify the stability of DNA strand, raising the likelihood of non-disassociation between strands or nonspecific binding of probes to unintended sites. Additionally, high-GC regions might form secondary structures that can interfere with capture or sequencing. Conversely, extremely low GC ratios weaken probe-target DNA binding, potentially leading to probe detachment.
Typically, these sequence features are more prevalent in introns or intergenic regions compared to exonic regions. Hence, panels encompassing more intronic or intergenic regions might exhibit lower efficiency compared to those that do not. Celemics conducts various researches and validation studies to enhance panel performance in these regions.