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Old 06-23-2019, 04:32 AM   #3
alextheinnes
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Location: Scotland

Join Date: Nov 2017
Posts: 3
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Hi SNPsaurus,

Thanks for the response and apologies for the delayed reply. Ive been away from the computer.

They did add PhiX yes at 5% to compensate for the low complexity region. The cut sites are for MseI and PstI. Following ligation to the adapters the cut sites should GGTAA for MseI and TGCAG for PstI. Both appear to have been affected equally by the quality drop in FastQC and the loss of specificity in adapter ligation. The size selection if anything was tighter this time round so i would have expected better coverage. I have yet to try mapping the reads to see how far they sit from the expected cut sites.

Ive now talked further with the sequencing facility and they have explained that the overclustering issue was caused by high levels of polyclonal or multi-occupancy wells. The automatic filters then remove those wells from further sequencing resulting in a much lower final read count than expected. What they are not sure about is why there are such high levels of multi-occupancy with this library.

With regards to the low quality cut site signal in the fastQC files, they suggest that this is usual for ddRAd and other Rad based sequence libraries where the low complexity region results in lower call confidence values from the sequencer. What is strange about this is that the PhiX should have negated this issue and that this was not observed at all in the first library.

The question now is whether the multi-occupancy levels, the low quality cut site call confidence, and the poor adapter specificity are related in any way. My initial thought was the low quality score and the adapter specificity must be, but on second thought perhaps not. If the overhangs had been blunted and I had the blunt ends ligating to random sheared fragments Id have thought they should still have formed coherent fragments for sequencing? Could the base pairs around the blunt end ligation region be degraded in some way that would affect the call confidence during sequencing?

Thanks again for the help and insight.

Alex
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