I am carrying out circular chromosome conformation capture (4C), largely following the van de Werken protocol.
I've amplified my libraries using pairs of primers specific to different viewpoints. These are all sequence-specific primers with Illumina sequencing adapters on the 5' end.
I was pointed to these adapters by technical support on page 10 of the customer sequence letter - they are under TruSeq HT Sample Prep Kits. I was told by another contact that these are the "universal" adapters that should work on MiSeq and HiSeq. The sequences are below:
AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT - read adapter
CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC - nonread adapter
Amplification with the adapter-tagged primers appeared successful when running the products on a gel, and analysis on the Bioanalyzer indicated that I had a peak of a few hundred bp for each library (with some DNA at higher and lower sizes).
However, when I sequenced on the MiSeq, no clusters were detected. It doesn't seem to be a machine issue - I've been advised that it's probably the custom primers or the libraries.
I am going to do qPCR with NEBNext Library Quant Kit to see if the adapters are present (I believe this should work, even with my longer adapters?). But does anyone have any idea what might have done wrong here? Are my adapters wrong?
Thanks!
I've amplified my libraries using pairs of primers specific to different viewpoints. These are all sequence-specific primers with Illumina sequencing adapters on the 5' end.
I was pointed to these adapters by technical support on page 10 of the customer sequence letter - they are under TruSeq HT Sample Prep Kits. I was told by another contact that these are the "universal" adapters that should work on MiSeq and HiSeq. The sequences are below:
AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT - read adapter
CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC - nonread adapter
Amplification with the adapter-tagged primers appeared successful when running the products on a gel, and analysis on the Bioanalyzer indicated that I had a peak of a few hundred bp for each library (with some DNA at higher and lower sizes).
However, when I sequenced on the MiSeq, no clusters were detected. It doesn't seem to be a machine issue - I've been advised that it's probably the custom primers or the libraries.
I am going to do qPCR with NEBNext Library Quant Kit to see if the adapters are present (I believe this should work, even with my longer adapters?). But does anyone have any idea what might have done wrong here? Are my adapters wrong?
Thanks!
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