I am trying to build a NGS library on enriched DNA (DNA length = 93 bp). I am using PCR amplification with two overhanging primers, both containing Illumina adapter sequences (Universal Adapter & TruSeq Adapter w/ barcode). My final library should be (~182 bp):

Universal Adapter (-), Enriched DNA (+), TruSeq Adapter(=)


After PCR amplification and QC by gel, I am seeing fragments of larger size. Analysis by Bioanalyzer indicates that I am might be getting some polymerization of the TruSeq adapter on the library. This is most likely due to bad primer design, with the primer containing two recognition sequences (21-nt), one at the 3' end and one at the 5' end of the TruSeq adapter

My question is:

How would the NGS analysis be affected if a library is submitting for sequencing, containing a mixture of single and dbl TruSeq adaptors?


Universal Adapter (-), Enriched DNA (+), TruSeq Adapter(=)



Universal Adapter (-), Enriched DNA (+), TruSeq Adapter 1(=), TruSeq Adapter 2(=)


Would the sequencing info from the "double library" be lost or would those fragments be read, just fine?


  • $\begingroup$ Remember to upvote answers you find useful. :-) $\endgroup$ – Forest May 28 '17 at 16:15

In short, you stand to lose information from your library due to the length of the fragments you're sequencing.

When you set up your sequencing run, you'll set the parameter for the number of sequencing cycles, which will determine the length of the reads to be sequenced. The longer the fragments contained in your library, the less they'll be fully sequenced.

A good idea would be to sequence your suspected polymerized fragments to verify that primer polymerization is really occurring. Just send your sample for normal sequencing, as you would for plasmids and PCR fragments.

If your primers are polymerizing because of inefficient design, you should fix this before loading your library into the Illumina device. If the primer-dimer effect comprises a significant portion of your library, this will only get amplified during the run and can definitely lead to a significantly less complex library. Sequencing a low-complexity library is a really expensive mistake whose one guaranteed result is to enrage your advisor and make him/her loudly question your competence. Trust me.

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