Hello,
I get data from the local sequencing facility, where they use nextera and duel indexing in a MiSeq machine.
I have some issues with multiplexed samples, where sometimes a lot of sequence goes to the unassigned pool. Most often 1 sample is completely missing and the amount of sequence in the unassigned pool roughly corresponds to what I expect.
I looked in the appropriate report files (namely DemultiplexSummaryF1L1.txt), and all the expected indexes (both 1 and 2) are the vast majority of the readings (though I'm kind of surprised with the enormous amount of variants in the indexes - in just 8 bases!).
Thus I don't think it is an incorrect index in the configuration file, otherwise other samples would also be affected. Since two times in a row it was the same combination of indexes that failed, I thought that might be the problem, but in subsequent runs the combination changed... so I'm left trying to figure out what is going on.
I wanted to go back and try the bascalling myself, but so far couldn't see of a way of doing it easily in MiSeq other than by the MiSeq Reporter...
I tried bcl2fastq and after quite some time fighting to get it compiled, I couldn't make it work with MiSeq data: file name extensions and versions are apparently not adequate for it.
Any ideas welcome.
Thanks,
Daniel
I get data from the local sequencing facility, where they use nextera and duel indexing in a MiSeq machine.
I have some issues with multiplexed samples, where sometimes a lot of sequence goes to the unassigned pool. Most often 1 sample is completely missing and the amount of sequence in the unassigned pool roughly corresponds to what I expect.
I looked in the appropriate report files (namely DemultiplexSummaryF1L1.txt), and all the expected indexes (both 1 and 2) are the vast majority of the readings (though I'm kind of surprised with the enormous amount of variants in the indexes - in just 8 bases!).
Thus I don't think it is an incorrect index in the configuration file, otherwise other samples would also be affected. Since two times in a row it was the same combination of indexes that failed, I thought that might be the problem, but in subsequent runs the combination changed... so I'm left trying to figure out what is going on.
I wanted to go back and try the bascalling myself, but so far couldn't see of a way of doing it easily in MiSeq other than by the MiSeq Reporter...
I tried bcl2fastq and after quite some time fighting to get it compiled, I couldn't make it work with MiSeq data: file name extensions and versions are apparently not adequate for it.
Any ideas welcome.
Thanks,
Daniel
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