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Library with strong secondary structures
What is known about the strand displacement activity of the DNA polymerase Illumina uses for bridge amplification? I have a library with strong secondary structures and I am worried that clusters do not form well due to the secondary structures. Does anyone have any tips to improve cluster formation for such a library?
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Anecdotally we find MiSeq to be the best sequencer for odd samples. You best bet is to sequence this library there. You can always a run a nano flowcell at a much reduced cost to test before you run full library. -
You can also try a 95C denaturation (instead of the usual NaOH denaturation). 1 minute at that temperature (in RSB or EB should be fine), just make sure you have a decent volume there so you don't evaporate everything. TruSeq Custom Amplicon product does this method to make the libraries nice and single stranded for cluster generation.Comment
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Amplicon sequencing is a targeted approach that allows researchers to investigate specific regions of the genome. This technique is routinely used in applications such as variant identification, clinical research, and infectious disease surveillance. The amplicon sequencing process begins by designing primers that flank the regions of interest. The DNA sequences are then amplified through PCR (typically multiplex PCR) to produce amplicons complementary to the targets. RNA targets...03-21-2023, 01:49 PM -
Targeted sequencing is an effective way to sequence and analyze specific genomic regions of interest. This method enables researchers to focus their efforts on their desired targets, as opposed to other methods like whole genome sequencing that involve the sequencing of total DNA. Utilizing targeted sequencing is an attractive option for many researchers because it is often faster, more cost-effective, and only generates applicable data. While there are many approaches...03-10-2023, 05:31 AM
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