TruSeq RNA kit adaptor problem

[yhuang1]

Hi, I am having problem with the TruSeq RNA kit lately - some of the adaptors totally failed and show funny "ladder-like" bands after PCR amplification (attached pic 1-2 show traces, pic 6 show virtual gel). Others seems to have worked and created a library but the peak looks rather odd and having "spikes" (attached pic 4-5). Has anyone encountered similar problem and figured out what was wrong? Any idea is appreciated!

[pmiguel]

Hi, I am having problem with the TruSeq RNA kit lately - some of the adaptors totally failed and show funny "ladder-like" bands after PCR amplification (attached pic 1-2 show traces, pic 6 show virtual gel). Others seems to have worked and created a library but the peak looks rather odd and having "spikes" (attached pic 4-5). Has anyone encountered similar problem and figured out what was wrong? Any idea is appreciated!

TruSeq kits offer the option to spike in control DNAs. Not sure if the sizes match your "ladders", but seems plausible. Did you use the control DNA?

--
Phillip

[yhuang1]

Thanks for the reply Philip -

I did not use the spike in control. I never thought they are very useful - you find out what was wrong after the sequencing - if you are able to get to the sequencing step, that means the library prep must have been successful anyway.

My own guess of the ladder-like bands and spiky peaks is that they are adaptors which somehow ligated to themselves. Different molecular weight probably represent different copies of self-ligated adaptors. But it is just a guess. I was hoping to see if someone has similar problem and already found out the answer.

Yanmei

[epistatic]

We do several hundred RNA libraries a month and we had this same issue pop up with only a few adapters in a kit, not all. We were able to show it was not sample related by having the same starting sample split and made into different libraries to look at possible index bias. Illumina said it was an adapter quality problem and replaced the RNA-seq kits.

[yhuang1]

Thanks a lot for the information!! I indeed strongly believe the problem came from the kit and most likely the adaptors. My starting RNAs are of great quality based on bioanalyzer. I myself have used up one kit before and in that kit only two of the adaptors had the problem. I actually ordered oligos from IDT and made the adaptor myself and it worked. However, this time almost half of 12 adaptors are completely bad and the other 5 gave "spiky" products. Only one adaptor produced a library with nice, smooth peak in bioanalyzer. It would cost us a fortune to remake all those adaptors ourself. It is good to know that Illumina would replace the bad kit. But even if they do, it is still a terrible thing that the users wasted a tremendous amount of time and precious samples!

[weigrc]

Hello yhuang1 and epistatic,

I am a bit scared after this thread since we are going to use TruSeq RNA prep kit. Would you mind to advise on which adaptor(s) may have this issue. Much appreciated!!!

[yhuang1]

There is no way to know beforehand which adaptor might have the problem because the problem did not come from the adaptor sequence. I have used the same adaptor (for example, index 2) from two different kits; one worked well while the other failed. It is probably a random quality issue. If your sample is precious, you probably should try the whole procedure on some other easy-to-get RNA samples first. In my experience, once you find out one adaptor works, it works every time. The failing adaptors fail everytime on all different RNA samples I have tried (that's another reason why I am very sure that the problem did not come from my RNA).

[weigrc]

Thank you for the information and just can wish to have the good luck in this lottery~~~

[danielr]

Thanks for the reply Philip -
...if you are able to get to the sequencing step, that means the library prep must have been successful anyway.

Not quite. The sequencing can seem to go just fine until you look at the output and just see a lot of the same adapter sequence. That tends to happen when we sequence those sawtooth pattern libraries.