My on-target reads seem to be lower, about 80%, with this 86kb target panel. With our 35kb panel, the on-target reads were about 95%. We are using ampliseq and to make matters more complex when we barcode just 2 samples of the 86kb panel our on-target goes down to about 60%. Any sugesstions to improve the data? Thanks.
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Hi there,Originally posted by cmccabe View Post
In our experience, off-target reads are typically a combination of specific per panel and per sample effects. For example, the best HMW DNAs will tend to have very similar off-target rate, while FFPE DNA - particularly when very degraded, can yield higher off-target rates, and each panel will have an off-target contribution based on the specificity of the primers as well.
There is no reason why multiplexing two or more samples on one chip should be expected to increase off-target rate overall. If you are seeing an increase in off-target between samples with the same primer panel it could be sample specific. If you haven't done so already, I'd recommend a follow up with your Field Applications Scientist (FAS) to ensure there aren't other confounding factors that are at issue. They will be able to dig into the full set of data and offer guidance. -
on-target reads
I'm thinking maybe it is sample specific as for 6 runs the same pattern has been consistent. The NA12878 cell line has alsway given lower on-target reads (~50%), then the NS12911 (gDNA) sample which has an on-target ~86%. We are goig to try two gDNA samples to confirm. Thanks.Comment
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