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-   -   Primer dimer sequence (http://seqanswers.com/forums/showthread.php?t=79941)

ericoml 01-03-2018 07:05 AM

Primer dimer sequence
 
Hi

I've just got sequence result from Hiseq 2500, single-end 100bp run with Riboseq samples. I've notice little adapter dimer contamination in the final library which is hard to get free with this kind of library. I confirmed this by Bioanalyzer, which shows a very small peak at 120 bp relative to adapter dimer and a 170 bp peak from the riboseq library.

In the end 8% of my reads are my 3' primer adapter, which I think the source is the dimer contamination.

However, I've notice something interesting in these sequences: after the 3' adapter sequence end, there is a common sequence comprising of an A stretch, which extend to the end of the read, like this one:

GAAAAAAAAAAAACAGACAAACACCTACATCGACACACGACGT

I was wandering the origin of this sequence, since it did not match my peaks on Bioanalyzer, neither were introduced my experimental steps. The 120 bp adapter dimer should be the end of the read, however the above sequence is appearing in my reads. Someone with adapter dimer problems noticed something similar?

My hypothesis is that this is relative to some flow cell Oligonucleotide attachment sequence upstream to the P5/P7 adapter binding site. I think this is being sequenced since my 3' adapter is only 60 bases, so this can be from some flow cell oligo. I try to search the flow cell oligo sequence but I was unable to find.

Best

nucacidhunter 01-03-2018 12:35 PM

A stretch of A homopolymer after reading through adapters is from immobilized oligo C and D on flow cell but it should appear at the 3' end of sequence. In your example it is at 5' end. Look at post #6 in the following thread:

http://seqanswers.com/forums/showthread.php?t=66237

ericoml 01-04-2018 12:09 AM

Thanks.

This sequence indeed appears at the 3' end of my reads. I believe you thought it was at 5' but this is because I cut the remaining sequences. One example of the full lenght adapter dimer read:

GAAGNGCACACGTCTGAACTCCAGTCACGCAGCTATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAGACTACACATAATGCGACACGATAGAAC

I was concerned that the sequence was artificially introduced in the experimental steps. Now I'm convinced that it's related to sequencing of the immobilized oligos.


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