Hi all,
I am using IDT capture probe to enrich the target region of tumor samples for sequencing.
However, I found Skewed GC bias in NGS data, which resulted in poor capture uniformity and lower depth of coverage, especially for those ctDNA samples, which is shown in the attached figure.
I tried gradient PCR, but not help a lot.
IDT suggested using cooler wash temperature to increase the enrichment of lower GC region. However, this will largely decrease the capture rate across the genome.
Can anyone give me some suggestion.
Thanks,
Junfeng
Hi Junfeng Jiang
GC biases is always viewed in molecular biology methods, even in NGS.The best way to avoid this problem is the addition of DMSO or betaine (no more than 5% of the total volume). These molecules will decrease the impact of GC bounds higher than AT ones on secondary structures and therefore increase the accessibility of molecules as primers and enzymes.
fred
Frederic Lepretre
Hi Fred,
Thanks for reply.
DMSO or betaine can actually avoid the GC bias, especially for those GC-rish region, which have been usually applied in PCR reactions.
I am wondering if it can work in the hybridization protocol?
Besides, which step should be involved? Library preparation or hybridization?
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