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Old 02-06-2012, 06:05 AM   #1
ZAAB
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Default 50+% of my HiSEQ reads are 3' primer (custom primer used)

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.
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Old 02-06-2012, 07:54 AM   #2
NextGenSeq
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Did you synthesize your adapters with phosphothioate bonds in between the ends of the last two 3' bases?

This prevents exonuclease digestion and adapter artifacts.
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Old 02-06-2012, 07:57 AM   #3
ZAAB
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My PCR primers for generating the construct BEFORE illumina adapter ligation are just regular primers.
I used the Illumina DNA sample prep kit, so the primers were Illumina, and hence, should have had that O-bond.
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Old 02-06-2012, 11:00 AM   #4
NextGenSeq
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I would redesign my PCR primers and cut down on the concentration. If you still get primer artifacts gel purify before adapter ligation.

I assume you purified by some method before ligating the adapters? If not there will be PCR primers with your product.

Also, don't design the primers with an A at the end since the T overhang from the adaptor might bind to it.
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