Hi all,
I've just had a faulty MiSeq run and before I re-run my samples, I'd like to make sure the next run is going to be valid, especially since it's a rather expensive technique. It'd be great if you could help me with your experience and advise.
Background of the samples and the run: The samples constitute gel-purified PCR products of a segment of a gene (270bp), which contains 15 randomized positions. Illumina adapters were 'ligated' to the samples by PCR, one primer containing multiplex barcodes, the other containing random barcodes (NNNN). Sample concentrations were quantified with the KAPA qPCR kit.
For the sequencing run, 16 pM of sample were loaded with a 1% spike of PhiX and 2x151bp were sequenced. These are the run's primary analysis plots from BaseSpace (secondary analysis hasn't yet been done):
The run returned ca. 50 reads on one paired end, 104'000 from the reverse. This is especially striking because the same samples have been measured previously in a 2x36bp run with 80% PhiX and 600'000 reads for my samples were obtained.
The possible errors that I've identified are the following:
1) I've overloaded the flow cell. I've used 16 pM sample, thus I'm on the top end of the recommended amount. In addition, the KAPA qPCR in retrospect was not measured in the linear 15-25 cycle range, but rather around 10-15 cycles. I may have underestimated my concentration up to a factor 1.8-2. If this is the error, I'm happy to know it and rerun with less sample.
2) The PhiX tech note says that in the first four cycles it is essential for the machine to have a complex library with an equal representation of the four bases. In addition, they say that for low complexity libraries, more PhiX is recommended. They say up to 40% (seems a waste to me, but if it's necessary can be done).
3) Looking at the log files, I get at times a 'Piezo' error and 'Position error before settling'. Does this characterize a specific type of failure?
4) After cloning my samples into a CloneJet vector and sequencing 20 colonies I've realized that many of the Illumina adapters appear truncated. Thus I've now ordered HPLC-purified primers to improve their quality.
Did I miss anything here? Are there additional error sources I need to consider? I'm happy for all your inputs.
Thanks,
Simon
I've just had a faulty MiSeq run and before I re-run my samples, I'd like to make sure the next run is going to be valid, especially since it's a rather expensive technique. It'd be great if you could help me with your experience and advise.
Background of the samples and the run: The samples constitute gel-purified PCR products of a segment of a gene (270bp), which contains 15 randomized positions. Illumina adapters were 'ligated' to the samples by PCR, one primer containing multiplex barcodes, the other containing random barcodes (NNNN). Sample concentrations were quantified with the KAPA qPCR kit.
For the sequencing run, 16 pM of sample were loaded with a 1% spike of PhiX and 2x151bp were sequenced. These are the run's primary analysis plots from BaseSpace (secondary analysis hasn't yet been done):
The run returned ca. 50 reads on one paired end, 104'000 from the reverse. This is especially striking because the same samples have been measured previously in a 2x36bp run with 80% PhiX and 600'000 reads for my samples were obtained.
The possible errors that I've identified are the following:
1) I've overloaded the flow cell. I've used 16 pM sample, thus I'm on the top end of the recommended amount. In addition, the KAPA qPCR in retrospect was not measured in the linear 15-25 cycle range, but rather around 10-15 cycles. I may have underestimated my concentration up to a factor 1.8-2. If this is the error, I'm happy to know it and rerun with less sample.
2) The PhiX tech note says that in the first four cycles it is essential for the machine to have a complex library with an equal representation of the four bases. In addition, they say that for low complexity libraries, more PhiX is recommended. They say up to 40% (seems a waste to me, but if it's necessary can be done).
3) Looking at the log files, I get at times a 'Piezo' error and 'Position error before settling'. Does this characterize a specific type of failure?
4) After cloning my samples into a CloneJet vector and sequencing 20 colonies I've realized that many of the Illumina adapters appear truncated. Thus I've now ordered HPLC-purified primers to improve their quality.
Did I miss anything here? Are there additional error sources I need to consider? I'm happy for all your inputs.
Thanks,
Simon
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