Hello everyone,
I was dealing with library construction for low complexity region from human genome,
And I also found that, the sequencing depth are obviously bias: which means, some reads were sequenced more than 100X, even more than 1000X, but other reads were sequenced only no more than 1X, I want to know why? and how to fix it.
Thank you for your attention.
Look forward to your answer.
Hi Qinglan,
there are few technologies to run NGS, but librairies preparation turn always around amplification and capture. some bias are revealed from both enrichments but the biggest is from amplification which is really dependent on GC contents and low complexity sequences. Most of us that tried both had better results with capture (from Agilent or Nimblegen or Nextera) with a better repartition and homogeneity of depth.
hope that helps
fred
Frederic Lepretre Hi, Frederic, sorry, I don't understand. You mean that I could try to use amplification reaction conditions that used to amply high of low GC contents sequences?
amplification as by amplicon from agilent, no, but capture as in this example (https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-rapid-capture-custom-enrichment.html). it should be better for you.
fred
Frederic Lepretre aha, I understood, thank you, fred. But I have already used capture, and the depth bias still there. sad
Frederic Lepretre And, fred, I am afraid I have to ask, will you add spike-in control when you doing capture sequencing? Will it be helpful if I add spike in controls?
ok Qinglan Li , one other tip that could help you is to ensure of the best quality of your samples. the higher it will be, the finest the capture will be. do you shear your samples for library preparation? if yes, advanced shearing (too much) could enable preparation.
fred
Frederic Lepretre Yep, I sheared my sample with best parameters, I checked the bands by agarose gel after shear, this step may not be the reason.
ok, you were talking about low complexity in human genome. what is it? centromeres or telomeres, or family genes or repeats...?
fred
oh, repeats...
maybe you should think on longPCR that can be sheared and sequenced with UDI or barcodes?
fred
Frederic Lepretre ok, thank you so much fred, I will think about your advice by looking more information.
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