Neb library preparation and sigma ordered adapters

[mchotalia]

Hi,

I have trying to combine the NEBNext sample prep kit with Sigma ordered Paired-end primers and adapters to prepare paired-ended libraries of genomic DNA....but i am having problems.

I recently made libraries with genomic DNA and observed a strange band at 120-150bp..this isn't primer/dimers as they run much lower nor the library. I do not do a size selection before the enrichment but after.

I tried to TA clone the band and observed lots of positive colonies and few negative ones. However, when i look at the results only a 5bp product has ligated (very weird).

Any ideas would be much appreciated.

Thanks

[pmiguel]

How about:

The 120-150 bp band is really a 240-300 nucleotide single-stranded DNA band. This is not clonable at all -- so your TA "clones" are just background vector prep contaminants.

(Did you do a "no insert" control for the TA cloning?)

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Phillip

[sc10021]

hi pmiguel

can you elaborate on the band and what it is please? I see it in my RNA and DNA preps as well
TQ

[pmiguel]

Hi sc10021,

That was just a wild guess based on the very limited information you provided. Perhaps you could expand on what you expected to see. Also what you mean by "RNA and DNA preps". Libraries? "Preps" to me, means sample prior to any processing.

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Phillip

[mchotalia]

Hi Phillip,

Many thanks for your response.

When i TA cloned the band i had very few colonies on my 'on template' control. In addition, the sequencing results show insertions of 5 bps, which clearly doesn't make sense since the ligation were set up with a 150bp band. I don't quite understand your response to my question would you mind explaining it further?

I called Illumina yesterday and they said the band is likely to be unused adapters that are PCR'ed in the enrichment. The rep said that many people dilute the adapters prior to the ligation step in their protocol. Do you have experience with this?

Thanks

[pmiguel]

Hi mchotalia,

We haven't made Illumina libraries yet. Only SOLiD and 454 ones. That said, I was looking at 4 Illumina libraries that we were given on the bioanalyzer. I thought one had your 150 bp bands! But it turned out just to be a glitch--another run of the same sample did not show it...

As to what I meant: During PCR amplification of a library you could end up with a situation where the reaction ran out of one of the primers. The reaction would continue to amplify one of the strands, but not the other. The result would be a certain amount of single stranded DNA. This would still show up on an agarose gel. Dyes like EtBr do no fluoresce as strongly when interacting single stranded molecules, but you do still see them.

Because the ssDNA molecule would be 1/2 the molecular weight of the double stranded version it would run as if it were roughly 1/2 the "size".

If you isolated this ssDNA, it would not be "clonable" via normal TA cloning method--that is for dsDNA with 3' A-overhangs. But if you used no insert at all, it is possible that your TA cloning vector would have some "background" vector contamination. These might be the 5-bp insert "clones" you are seeing. They may, effectively, be "uncut vector".

As I mentioned above, this is all blind guess-work on my part. I don't even know what TA cloning method you are using, or which library protocol or kit you are using to construct Illumina libraries. You may not even be using PCR. (If I remember correctly the new Trueseq protocols do not require it?)

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Phillip

[NextGenSeq]

The 120 bp artifact is well known and has been posted about multiple times. We found that reducing primer concentrations you can get mostly rid of it.

[pmiguel]

Is it an adaptor dimer? That is, if you do not get rid of it, do you get a bunch of reads with no inserts?

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Phillip