I have developed this multiplex PCR panel for next generation sequencing library prepration. This panel is used for the diagnosis of a particular bacteria infection, as well as some SNP I'm interested in.
The panel works well with sputum samples, but failed to detected some expected SNPs when we tested with FFPE samples. The copy number of this bacteria might be lower (120) than my detection limit (250). We still managed to get at least 5000 coverage in most of the SNP locations. But only about half the SNPs were called, why not the others?
Hi Jialiang Lin
amplicons panels need to be sure samples are in good shape and designed amplicons not too long. dis you test quality of your samples (DIN or RIN) and how long are your amplicons?
fred
Thank you Frederic Lepretre for the suggestion.
No, we didn't analyse the DINs of the DNA samples I'm afraid. The amplicons are on average 150 bp long. I understand you might be wondering if the DNAs were too fragmented, in which case might cause the drop in reads and the mutation rate subsequently. But in fact, I saw at least 5000 coverage in almost every amplicon for this particular sample, and they are filtered reads. There wouldn't be this many reads if there's fragmentation right?
J
yep
fragmentation could generate small fragments that can't match with amplicons length, droping down the coverage on some of your targets, and when you add GC influence on amplification...
all the best
fred
Frederic Lepretre
Hi,
I have the same problem with ARMS PCR to detect SNPs.
I have developed a panel to detect Helicobacter pylori's levofloxacin and clarithromycin resistant genes and used this primer probes:
Article Evaluation of multiplex ARMS-PCR for detection of Helicobact...
I have amplification in 4 out of 5 primer probes designed for levofloxacin resistant gene SNPs when I test ATCC strains.
I have amplification for clarithromycin resistant gene(A2142C) SNP in all samples and ATCC controls.
I have used ATCC-43504 and ATCC-700392 (26695) as the reference which are negative for levofloxacin and clarithromycin resistant genes. and all samples are FFPE stomach positive samples.
Is that for DNA fragmentation during extraction?
You can see my screenshots here:
https://www.researchgate.net/post/An_issue_during_amplification_of_Hpylori_resistant_genes_A2142C_GyrA271T_GyrA272G_GyrA261G_and_GyrA271A
Hi Hesam
FFPE samples are always degraded, the problem is to check how much they are.
can you test RIN or DIN to check it?
degradation will create lower length templates, not enough long to get enough template to run RCR in good conditions and introduce a bias in targets representations along samples.
all the best
fred
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