I was reading the ScriptSeq Index PCR Primers (1-12) protocol, and it says:
Since the Index sequence of pooled, multiplexed ScriptSeq libraries will be read simultaneously, it is important to maintain color balance for each base of the Index sequences in the pooled library. Otherwise, Index sequencing will fail due to registration failure.
Then I'm thinking how will this affect the sequencing? Could the registration failure lead to an incorrect barcode assignment? Or will the read just be assigned some nonsense sequence?
Also why is it important that the short barcodes match up for the red and green laser? What about the entire read length, shouldn't they also "match" up then?
The reason I ask is that i have RNA seq data from a six-pooled Illumina run using indexes that apparently should not have be mixed
Any inputs are appreciated.
Since the Index sequence of pooled, multiplexed ScriptSeq libraries will be read simultaneously, it is important to maintain color balance for each base of the Index sequences in the pooled library. Otherwise, Index sequencing will fail due to registration failure.
Then I'm thinking how will this affect the sequencing? Could the registration failure lead to an incorrect barcode assignment? Or will the read just be assigned some nonsense sequence?
Also why is it important that the short barcodes match up for the red and green laser? What about the entire read length, shouldn't they also "match" up then?
The reason I ask is that i have RNA seq data from a six-pooled Illumina run using indexes that apparently should not have be mixed
Any inputs are appreciated.
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