I have sequenced numerous multiplexed pools of BS amplicon-seq libraries derived from human samples on a MiSeq over the past few weeks. I have been utilising trim-galore and Bismark for alignment and am finding the mapping efficiency to be highly variable across pools:
pool1 - ~85% - no PhiX spiked in
pool2 - ~70% - no PhiX spiked-in
pool3 - ~55% - 10% PhiX spiked-in
pool4 - ~30% - 10% PhiX spiked-in
How do I go about trouble shooting this? Which factors are likely to affect mapping efficiency?
The MiSeq run metrics were all very ideal for all of these pools, so I don't think anything strange happened on the sequencing side of things. The amplicons were around 135bp before transformation to sequencing libraries and the later two pools (pool3 and pool4) are between 100-135bp.
If any additional info is needed then please do ask.
Edit:
I forgot to look at the fastqc files for the last two pools. Quickly looked at them and most of the per base sequence content for cytosine hovers around 10-20% throughout the entire read. I assume this means that bisulfite conversion was unsuccessful before library construction? This would likely affect the mapping efficiency?
pool1 - ~85% - no PhiX spiked in
pool2 - ~70% - no PhiX spiked-in
pool3 - ~55% - 10% PhiX spiked-in
pool4 - ~30% - 10% PhiX spiked-in
How do I go about trouble shooting this? Which factors are likely to affect mapping efficiency?
The MiSeq run metrics were all very ideal for all of these pools, so I don't think anything strange happened on the sequencing side of things. The amplicons were around 135bp before transformation to sequencing libraries and the later two pools (pool3 and pool4) are between 100-135bp.
If any additional info is needed then please do ask.
Edit:
I forgot to look at the fastqc files for the last two pools. Quickly looked at them and most of the per base sequence content for cytosine hovers around 10-20% throughout the entire read. I assume this means that bisulfite conversion was unsuccessful before library construction? This would likely affect the mapping efficiency?
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