Hello,
I have data output from low complexity ampseq run. The read counts are very low (<100 reads per well) and the spike-ins% is very high (>80%) across all of the samples. We checked QC for the run and have not observed any issue with the run. I am not sure what could go wrong during experiment or library prep that contribute to this. Lab scientists repeated the library prep. and we got the same result. Any suggestion or idea on What might be the reason for such a high spike-ins and low read count appreciated.
My first instinct is DNAse contamination, which would impact both the input material and the spike-in. If the spike-in reads are very high, and amplicon reads very low; then the contamination occurred prior to spike-in; of reads are low across the board then the contamination happened during library prep.
However, unless equipment was heavily contaminated, or there's contamination in some reagent, this typically impacts a single library prep, and should not be repeatable.
Another possibility is that the primers did not anneal; and sometimes this can happen if there are multiple batches of primers being used for different experiments, and something was mislabeled. Alternatively the thermocycler could be broken; but it typically issues an error or warning if it thinks something is wrong with the temperature.
