Hi everyone,
We’ve done several attempts at sequencing a low diversity library on a NextSeq Mid Output v2.5 flow cell, following the recommendations from the provider of the library preparation kit. Unfortunately all samples failed QC for low coverage. We noticed a few things that we haven’t been able to explain:
1. 30% PhiX was added but we only got out ~5%. We have done several attempts at sequencing this kind of library, adding between 20-40% PhiX, but measured PhiX is always considerably lower than added. PhiX from the same batch was used before and after our run(s) with little discrepancy between added and measured PhiX.
2. The thumbnail image shows normal cluster density but when it comes to clusters that were read there’s a large difference between the top and the bottom of the flow cell with very few clusters read at the bottom.
3. There is a drop in quality around cycle 20-40.
For the last run the flow cell was loaded with 1.5 pM, cluster density was 133 k/mm2 and the error rate <1%. We have checked for primer dimer contamination in the library pool but did not find this. There was a discrepancy between the concentration measured with Qubit and qPCR (almost twice as high concentration with Qubit), but this was the same for the PhiX and the library pool, so it doesn’t explain low measured PhiX. The input was based on Qubit, which makes overloading of the flow cell unlikely. We’ve been in contact with Illumina tech support but haven’t managed to find an explanation to why things went wrong, and therefore also don’t know what adjustments to make when we resequence.
Any suggestions/ advice would be very much appreciated!
Thanks.
We’ve done several attempts at sequencing a low diversity library on a NextSeq Mid Output v2.5 flow cell, following the recommendations from the provider of the library preparation kit. Unfortunately all samples failed QC for low coverage. We noticed a few things that we haven’t been able to explain:
1. 30% PhiX was added but we only got out ~5%. We have done several attempts at sequencing this kind of library, adding between 20-40% PhiX, but measured PhiX is always considerably lower than added. PhiX from the same batch was used before and after our run(s) with little discrepancy between added and measured PhiX.
2. The thumbnail image shows normal cluster density but when it comes to clusters that were read there’s a large difference between the top and the bottom of the flow cell with very few clusters read at the bottom.
3. There is a drop in quality around cycle 20-40.
For the last run the flow cell was loaded with 1.5 pM, cluster density was 133 k/mm2 and the error rate <1%. We have checked for primer dimer contamination in the library pool but did not find this. There was a discrepancy between the concentration measured with Qubit and qPCR (almost twice as high concentration with Qubit), but this was the same for the PhiX and the library pool, so it doesn’t explain low measured PhiX. The input was based on Qubit, which makes overloading of the flow cell unlikely. We’ve been in contact with Illumina tech support but haven’t managed to find an explanation to why things went wrong, and therefore also don’t know what adjustments to make when we resequence.
Any suggestions/ advice would be very much appreciated!
Thanks.
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