Hello all!
Recently I ran a library on our lab's new Next Seq 500 and came across an interesting problem. While processing the data from the instrument, I noticed that one of my samples had an excess of 35 bp poly(N) reads...as in 40% (yikes!). From what I can tell this generally means adapter dimer, as the bcl2fastq tool masks adapters with "N" as well as reads below the default of 35 bases. None of my other samples had this problem.
We had previously ran the samples before pooling on an Agilent TapeStation, and noticed this particular sample had a high concentration of adapter dimer. So we ran the library on a Blue Pippin to select for a bp range of 200-500, and rechecked with the TapeStation to confirm the 120 bp peak was gone. Which it was.
So we proceeded with the run. And then I discovered this problem.
I then ran some of what I had left of the size selected library on both a regular agarose gel and a denaturing gel. A ~120 bp band showed up on the denaturing gel only!
My question is, has anyone run into this before or do they know if some secondary structure could be causing it? Where could this band be hiding?
Recently I ran a library on our lab's new Next Seq 500 and came across an interesting problem. While processing the data from the instrument, I noticed that one of my samples had an excess of 35 bp poly(N) reads...as in 40% (yikes!). From what I can tell this generally means adapter dimer, as the bcl2fastq tool masks adapters with "N" as well as reads below the default of 35 bases. None of my other samples had this problem.
We had previously ran the samples before pooling on an Agilent TapeStation, and noticed this particular sample had a high concentration of adapter dimer. So we ran the library on a Blue Pippin to select for a bp range of 200-500, and rechecked with the TapeStation to confirm the 120 bp peak was gone. Which it was.
So we proceeded with the run. And then I discovered this problem.
I then ran some of what I had left of the size selected library on both a regular agarose gel and a denaturing gel. A ~120 bp band showed up on the denaturing gel only!
My question is, has anyone run into this before or do they know if some secondary structure could be causing it? Where could this band be hiding?
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