only short fragments (90, 124bp) after amplification

[chicken]

Dear community,

I am new at RNA seq with Illumina and hope you can help me.
We use the MiSeq System with the TruSeq Stranded mRNA Sample Prep Kit. I do low throughput.
After the amplification step I do a BioAnalyzer analysis to check the sample before pooling.
The BioAnalyzer shows me 2 peaks, one at approximately 90bp and one at 124bp. Any suggestions what this could be? I think its adapter dimers but I really dont know how they occur? Does anyone had that problem previously??
Dont hesitate to ask further questions.

[cmbetts]

124 is almost certainly adapter dimers. I'm unfamiliar with the Illumina kit, but there's usually a step that involves diluting the adapter to be the appropriate concentration for the amount of insert present.
Also, how much RNA are you starting with? You might be having issues either from starting from too little material or with your mRNA enrichment method.

[microgirl123]

Do you have a peak for your library also or just unligated adapter/adapter dimer peaks?

[chicken]

Hey thanks for your suggestions. Now I am quite sure that the peaks show adapter dimers. Unfortunately my kit manual does not show the option to dilute the adapter. Is this not common in illumina kits? I am starting with 500ng of total RNA. Any suggestions about diluting Adapter??

[pmiguel]

The TruSeq kits do not include a protocol for titrating adapter concentrations. If you end up with only adapter dimers it just means that library construction failed. Contact [email protected] for help.

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Phillip