I have generated an Illumina Sequencing Library by nested PCR and size-selected from agarose gel. When measured with Nanodrop, the DNA is clean and of perfect quality. When sequenced with Sanger, the sequence of the illumina adaptors are complete and the library size is as we expected. When ran in a gel, the library has the expected size.

Nevertheless, when quantified by standard Illumina KAPPA qPCR Library quantification kit, it seems that only between 2-10% of the DNA detected by Qbit is detectable by qPCR. On top of this, when we do a Spike-in of our library in a HighOutput SR75 flow cell, in a HiSeq550, our library only retrieves 0,5-1% of the expected reads.

Has it ever happened to anyone before that an apparently intact and correct illumina sequencing library does not render even near the yield expected? Any ideas of what could be happening?

The library size is 606bp, which is a bit longer than usual but there are many articles published that sequence libraries of that size an larger.

Thank you in advance!