Hi All:
We've run 7 lanes of Illumina sequencing with the HiSEQ.
We're sequencing the middle of a PCR product...
We're using a custom sequencing primer to avoid the common 5' end of the PCR product.
In many of our lanes, we 'see' about 50% of the reads being the 3' end Illumina adapter (barcode side)...
If we were just using the common Illumina adapter primer (hybridizing to the 5' end), I'd say I have a huge Illumina primer-dimer issue...
BUT I am using a custom primer...
So how does this happen?
Did I get my forward, non-5'-phosphate PCR primer filled in when A-tailing (without, ligation of adapters has bad efficiency (Separate issue)), and then Illumina primers ligated to it...
IS this possible>?
OR Is there regular Illumina sequencing primer contamination in my runs, and the Illumina primer dimer is being sequenced...?
Or?
Thanks in advance.
We've run 7 lanes of Illumina sequencing with the HiSEQ.
We're sequencing the middle of a PCR product...
We're using a custom sequencing primer to avoid the common 5' end of the PCR product.
In many of our lanes, we 'see' about 50% of the reads being the 3' end Illumina adapter (barcode side)...
If we were just using the common Illumina adapter primer (hybridizing to the 5' end), I'd say I have a huge Illumina primer-dimer issue...
BUT I am using a custom primer...
So how does this happen?
Did I get my forward, non-5'-phosphate PCR primer filled in when A-tailing (without, ligation of adapters has bad efficiency (Separate issue)), and then Illumina primers ligated to it...
IS this possible>?
OR Is there regular Illumina sequencing primer contamination in my runs, and the Illumina primer dimer is being sequenced...?
Or?
Thanks in advance.
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