sample cleanup question

[AngA34]

Hi everyone,

Does anyone know if Exo/SAP PCR cleanup is compatible with MiSeq Illuina sequencing?

I am preparing some environmental DNA for MiSeq Illumina sequencing. I have done the first round of PCR with 16S bacterial primers which have overhangs that will be used by the sequencing facility to perform a second round of PCR where they would attach the Illumina adaptors. My question is, is ExoSAP PCR cleanup of this first round of PCR compatible with their 2nd round of PCR and the Illumina MiSeq platform? Is there anything in the PCR reaction (normal PCR reaction performed with 3%v/v DMSO) or in the Exo/SAP reaction, that could interfere with lib preparation or sequencing? Does anyone have experience with this?

Thank you

[nucacidhunter]

ExoSAP-IT will remove nucleotides and remaining primers. I see two potential issues. If the first PCR has primer dimers, they will not be removed which may cause issues in 2nd PCR (such as preferential amplification of dimers rather than target region). Also, depending on how much of 1st PCR product is used as template for 2nd PCR, it may disturb buffer composition which may affect PCR efficiency. Using ExoSAP-IT for library clean up (2nd PCR) will leave all the primer-dimers and small artefact fragments in, which will result in sequencing those rather than target fragments.

[cement_head]

I'm not so sure that primer dimers would be an issue - it seems to be able to clean-up Adaptors.

[cement_head]

I'm not so sure that primer dimers would be an issue - it seems to be able to clean-up Adaptors.

Nope, won't work for NGS - just spoke with them - they've tried and doesn't.