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Old 11-18-2011, 03:14 AM   #1
malikian
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Default NGS library preparation followd by sanger seqeunding

Helo there,

I have a t(9;22) fusion transcript where I know that the fusion gene on the 3' end is the ABL1 gene and I want to find out what is the partner gene on the 5' end. I know that using RACE PCR is the typical strategy of investigating such cases, but I had this idea that I would like to know if its feasible from the experts.

The idea is to use RNA-seq library preparation kit to add adaptors to my transcript and then sequence it using sanger sequencing rather than NGS using a forward primer complementary to the 5' adaptor and a reverse primer specific to a sequence in the ABL1 gene on the 3' end.

My questions are:
Is this too naive to think about?
If it is doable, which kit would be ideal?
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Old 11-18-2011, 04:07 AM   #2
pmiguel
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This is a recapitulation of 5' RACE using an RNAseq library prep kit. Right? I don't see a difference.

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Old 11-18-2011, 04:16 AM   #3
malikian
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Kind of. I'm not managing to get race to work, but since the library prep kits are optimized (I assume), they could save time trying to fiddle around with race. I'm looking for a more efficient method of tagging the 5'end with a known sequence so I could do a PCR.
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Old 11-18-2011, 04:46 AM   #4
pmiguel
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Doubtful an RNAseq library kit would be more efficient at 5' RACE than a 5' RACE kit.

There are kits for creating transcription start site enriched cDNA libraries that use the eukaryotic 5' cap. Not sure if these would work for a fusion transcript, though.

What 5' RACE method/kit are you using? Are you doing nested PCR?

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Old 11-18-2011, 05:00 AM   #5
malikian
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I've used Ambion FirstChoice ® RLM-RACE Kit. The positive controls the kit provides work nicely, however, with my sample which is not as concentrated as the control RNA, I lose almost all of it during the phenol chloroform and ethanol precipitation steps.
I've also used an Invitrogen kit, where I managed to get some sort of tailing, but 5'-tag annealing primer (F-primer) was not annealing specifically.
I'm trying now to develop my own protocol (almost similar to what the Invitrogen kit follows) and try different conditions (simply because I can’t keep buying kits since they are too expensive).

I was just wondering if using the RNA-seq library prep kit could save me the hassle. Particularly that I need to do this only on one sample and perhaps never again.
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Old 11-18-2011, 05:31 AM   #6
pmiguel
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My advice to all molecular biologists is not to use phenol chloroform/etoh purification unless you have a particular affinity for the method. In the hands of a bench god it will work perfectly every time. But there are so many caveats to their use, that it is to be avoided by anyone who hasn't trained in a zen molecular biology monastery since childhood. Substitute those steps with something else (eg, Zymo columns).

Also there are plenty of 5' RACE methods that do not require kits.

Do you have good results making RNAseq libraries? If so, sure, go for it. I would say go for either no ribo-depletion, or a ribosome-specific method (eg, ribo-zero). You will still probably need to do nested PCR if your transcript of interest is rare.

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Old 11-18-2011, 10:35 AM   #7
malikian
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Thank you Phillip

I thought of using a zymo kit to avoid the phenol chloroform step, but the kit says that phenol chloroform step is important to get rid of the de-capping enzyme used in the step before.

I think I'll keep trying with the invitrogen reagents for a while and also try the RNA library prep method. the problem is that I don't have any experience in library predation as yet. perhaps that's why I thought it should be a more straight forward thing to do.
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Old 11-20-2011, 03:46 AM   #8
dongzw
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Very informative discussion! Thank you, both

From my experiece, Invitrogen's 5´RACE System for Rapid Amplification of cDNA Ends, Version 2.0 is better than RLM-RACE kit, in detecting fusion transcripts
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