Hello,
2/3 of my amplicons are failing to produce many/any sequences after MiSeq amplicon sequencing and I am lost as to why this might be. Here is a brief overview of the protocols I am using:
I have recently designed primers to amplify 3 regions of the same gene and all three primer pairs produce clean, strong bands on a gel after PCR amplification. I have been doing one round of PCR with Q5 polymerase and then using ~3ul of this as a template in a second reaction with taq polymerase to produce A overhangs needed for future ligation of the MiSeq adaptors. The primers I have been using contain phosphate modifications.
My PCR products are then cleaned (using SureClean) before ligating MiSeq adaptors (and indexes) using the QIAseq FX library prep kit. Samples are cleaned once again (using AMPure beads) before Qubit quantification. All of my samples have a good concentration of DNA. They are then run on the MiSeq using a MiSeq Reagent nano Kit v2 (2x 250bp).
One of my amplicons produces many high quality reads and the other two do not. I did not pool my amplicons, they are run separately. I have done this protocol twice now and the same thing has happened both times. I don't think this is an issue with the quality filtering during processing of the sequences as it seems like there are very few/none of my target sequences in the fastq files upon manual inspection.
Does anyone know why I might be having issues MiSeq sequencing specific amplicons?
Thanks in advance!
If you are trying to amplify three regions of the same gene, they are basically three different targets in sequencing sense. And unless they are of same size, you should not pool them. And I guess there are differences in targets' sizes. As you have not provided any information related to the targets, nothing can be said further.
Abhijeet Singh thank you for your response. The three different targets were not pooled, they were run separately.
Raul Riesco I've considered this but as the samples have all been prepared in the same way at the same time I can't think why some of the amplicons would work and others not. As far as I'm aware, if my product has amplified during the taq round of PCR there must be A-tailing (?) and so don't see what could be going wrong with adaptor ligation for some amplicons. I have quantified individual samples and the pooled library and all was as expected but I did not do any QC. We do have a fragment analyser so that is a good suggestion, thank you.
There could be several reasons why some of your amplicons are not producing many/any sequences after MiSeq amplicon sequencing. Here are a few possibilities:
To troubleshoot the issue, you may want to re-evaluate the primer design, PCR conditions, DNA quality and quantity, library preparation, and sequencing run parameters for each amplicon.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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