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Old 12-21-2010, 11:23 AM   #1
MarineMan
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Location: Ireland

Join Date: Dec 2010
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Default Small RNA sequencing library: Help! Very Strange PCR products

Hi

Sorry about the long post, I guess I just need to explain a few things. I'm just creating a small RNA library in the usual manner, and its been successfull, but recently its all starting to go wrong and some very strange stuff has started to happen.

I am using a self assembled kit to prepare a small RNA sequencing library. Im approaching it in the exact same way as the Illumina protocol (not v1.5) with gel separation of ligation products etc... except I'm using the IDT linker 1 (pre-adeneylated) as the 3' adaptor and then inserting the rest of the Illumina 3' adaptor/primer sequence through a custom primer at the PCR stage. As a result of the extra IDT sequence after my small RNA before the Illumina sequences, the correct PCR product for my library is ~110bp. I've recently successfully created a dsDNA sequencing library, when I run it on a gel (only a small amount of it) I am seeing the expected 110bp product and an 87bp artefact.

In order to do sanger sequencing from this library (to validate the library before sending it off for sequencing)I used this 1ul of this PCR product as template for another an identical PCR reaction using a different DNA polymerase as the high fidelity polymerase recommended by Illumina dies not give A-overhangs needed for cloning. This PCR gave the same products, I cloned the mixture into JM109 cells and sequenced from plasmids from 40 colonys. The majority of the inserts were the 87bp artefacts but some the correct PCR product i.e. small RNAs flanked by the correct sequences for Illumina sequencing, but I need to get rid of the PCR artefacts before Illumina sequencing.

Strangely the PCR artefact is not a primer dimer from PCR as there is no over lap of the 2 primer sequences, instead it is a fusion of the 2 PCR primers head to tail. I have no idea how this could happen, unless its a result of to ligated adaptors from earlier stages getting into PCR. However, as non-ligated adaptors never encounter each other during the process (PAGE separation of ligation products) I do not know how this could have happened.

It gets stranger, repeating the second PCR again gave me a completely different PCR product, it was massive and hardly migrated from the well (the gel was fine as the ladder migrated normally). I repeated the PCR and got the correct products. Iím tried gel extraction on this PCR product (be fore trying it on my original library), it didnít work. Luckily I had saved most of this PCR reaction at -20deg, so I ran it on a gel again and seen the correct products again but fainter this time, and also accompanied by numerous other bands ALL LARGER.

This has happened me several times, i.e. either the repeat PCRs give huge PCR products or they give the correct ones but when I return to them as a source of material for gel extraction optimization, lots of larger bands have appeared. It makes absolutely NO SENSE. I've no reason to suspect my original library is like this too, but I dont want to do a gel extraction on it and risk loosing it all. I want to optimise the process on the newer PCRs derived from this.

Has anyone ever experienced this before / have any idea what is going on. Its very frustrating as there a lot of work involved to get this far and its stopping me from carrying out optimization of the gel extraction process.

Cheers
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Old 12-23-2010, 04:31 AM   #2
elaney_k
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Location: Dublin, Ireland

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Hi Marineman,

Have you tried to do a second gel isolation step where you cut out only the 110bp fragment of your library? You will loose a certain percentage of your library but should get rid of most of the artefacts, and is something that's routinely done where there are libraries with lots of primer-dimers in cases where the library prep can't be repeated e.g. where there's limited sample.

Larger size fragments during PCRs have been noticed with other libraries but are usually due to over-cycling during the PCR step. I would do the second gel extraction and then if you still want to test your library with cloning/sanger sequencing reduce the number of PCR cycles/primer input amount and see if that helps.

Elaine
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