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Hi everyone,
We have been working with the Miseq for some time to duplicate a targeted genomics protocol we have successfully run on the HiSeq. Our first obstacle was discovering that the MiSeq uses PE technology (we used SR for HiSeq). After discovering this and having our MiSeq software upgraded so that we could use custom primers, we had a control library systhesized that would: work with our custom primers; anneal to the flow cell; and had variable regions for the first 8 sequencing reads and for the index reads. The thought was that this library would help us to do accurate base calls as our library complexity was low for these initial experiments.
We have run this control with our library 2 times now: the first time 50/50 the control library and 1 validated clone we would expect to work on the MiSeq, and once with 5% control and the rest our more diverse library (although there are still less than 10 species expected). In both cases, we get AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAANAAAAAAAA
althoug it looks like we have good clustering images in all channels
So I have 2 questions:
What could be causing this? is it just the low complexity of our libraries?
Are there any other methods to reanalyze the images to obtain accurate base calls?
Any help would be greatly appreciated.
Thanks,
Tim
We have been working with the Miseq for some time to duplicate a targeted genomics protocol we have successfully run on the HiSeq. Our first obstacle was discovering that the MiSeq uses PE technology (we used SR for HiSeq). After discovering this and having our MiSeq software upgraded so that we could use custom primers, we had a control library systhesized that would: work with our custom primers; anneal to the flow cell; and had variable regions for the first 8 sequencing reads and for the index reads. The thought was that this library would help us to do accurate base calls as our library complexity was low for these initial experiments.
We have run this control with our library 2 times now: the first time 50/50 the control library and 1 validated clone we would expect to work on the MiSeq, and once with 5% control and the rest our more diverse library (although there are still less than 10 species expected). In both cases, we get AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAANAAAAAAAA
althoug it looks like we have good clustering images in all channels
So I have 2 questions:
What could be causing this? is it just the low complexity of our libraries?
Are there any other methods to reanalyze the images to obtain accurate base calls?
Any help would be greatly appreciated.
Thanks,
Tim
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