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Old 12-07-2015, 05:11 AM   #1
seq198
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Location: Germany

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Default Hedgehog shaped cDNA library

Hi there,

I would like to ask whether someone has ever experienced a similar problem with hexamer primed cDNA libraries?

We have prepared a cDNA library starting with high quality RNA from mice (RIN = 9). In the final QC, we loaded a fraction of the lib onto a fragment analyzer. As you can see in the attached pic, the lib resembles a hedgehog with peaks every 10bp. When we sequenced the lib, we found that the peaks consisted of a decamer (GTATATACTA). Sometime up to 40 of these decamers are present. Thus, it´s not an issue of the fragment analyzer.

We have really no idea what this could be. We see that for different samples, but it´s not consistent. Some days the lib looks perfect, the other day we get this shape.

I´m really looking forward to your opinions!
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Old 12-07-2015, 02:15 PM   #2
nucacidhunter
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This seems to be a rare observation. Providing more info such as workflow, input quantity, kit and posting full sequences of few of those decapolymer reads might give clues on what might be cause.
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Old 12-21-2015, 05:44 AM   #3
seq198
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We are following exactly NEB kit instructions (NEBNext® Ultra™ RNA Library Prep Kit for Illumina® and NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina), we start with 500 ng of total RNA. Anyone else seeing this?
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Old 09-16-2017, 05:17 AM   #4
vincr
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Hi all,

Has anyone found an explanation for this? We are having the same problem and can't seem to find an explanation.

We have Drop-seq libraries that we have prepared with the Nextera XT kit, they all have an exactly similar pattern of hedgehog-like spikes (see attachement).

We have tried to use a new Nextera kit but it does the same effect. The cDNA before library prep did not have these spikes.

I am wondering if it could be a bioanalyzer issue, so I cleaned the electrodes and ran a diagnostics but did not find anything suspicious.

Does anyone have any thoughts on this?
Many thanks!
Vince
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Old 09-18-2017, 08:12 AM   #5
pmiguel
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Usually it either means your library is "bottomed-out" or you have some spike-in controls of fixed length. By "bottomed-out" I mean amplified from a very limited pool of input amplicons.

As to what the spikes actually are, I always suspect they are some sort of adapter-dimer concatamers. But that is just speculation on my part.

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