Hi all,
I am new to MiSeq sequencing and it really frustrates me lately.
I had two MiSeq run recently and they all turned out to have about 20% adapter get sequenced. I've been using the TruSeq PCR-free DNA prep kit for library preparation. So the first time I followed the protocol for library prep ant it gave me 20% adapter sequenced. The second time I did an 8 cycle PCR enrichment for my library, followed by gel extraction of DNA fragments ranging from 500bp to 1500bp, hoping to remove all adapter dimers. However it gave me about 20% adapter sequence as well, which also leads to an uneven distribution of my libraries (percent reads pass filter for all samples ranging from 0.2% to 24%).
I wonder if anyone knows why this happens? We don't have bioanalyzer, so I only used qPCR for library quantification using KAPA kit. Also if anybody knows where I could find a commercial bioanalyzer? I would like to pay for them to run a couple of samples for me, in order to get a better MiSeq result.
Thank you!!!
I am new to MiSeq sequencing and it really frustrates me lately.
I had two MiSeq run recently and they all turned out to have about 20% adapter get sequenced. I've been using the TruSeq PCR-free DNA prep kit for library preparation. So the first time I followed the protocol for library prep ant it gave me 20% adapter sequenced. The second time I did an 8 cycle PCR enrichment for my library, followed by gel extraction of DNA fragments ranging from 500bp to 1500bp, hoping to remove all adapter dimers. However it gave me about 20% adapter sequence as well, which also leads to an uneven distribution of my libraries (percent reads pass filter for all samples ranging from 0.2% to 24%).
I wonder if anyone knows why this happens? We don't have bioanalyzer, so I only used qPCR for library quantification using KAPA kit. Also if anybody knows where I could find a commercial bioanalyzer? I would like to pay for them to run a couple of samples for me, in order to get a better MiSeq result.
Thank you!!!
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