SMART Concatemeres

[sylvain]

Hi everybody

A this time, we have done six ½ Titanium run of cDNA sequencing. We always use SMART (SMARTER kit from Clontech) cDNA preparation without normalization. In the last run, we obtain at least 50% of SMART adaptor concatemer sequence.

Of course, we have followed the Clontech protocol and verified the quality and the amount of our RNA

Is anybody has an hypothesis to explain how this concatemer occur and how to modify the SMART original protocol in order to avoid this problem.

Thanks a lot

[vanillasky]

What did your cDNA look like in the end? Did you run it on a high sens. chip?

[ZWB]

We find that the primary cause of concatamers (assuming the RNA is abundant and of high quality) is over amplification in the final PCR step. I would try decreasing the number of PCR cycles. Alternatively, if you have low quality or very small amounts of RNA you should decrease the primer concentration during cDNA synthesis.

[ecPDCNRA]

Concatemer synthesis from template switching oligo

FYI
The article shows the mechanism of concatemer synthesis from template switching oligo. Please see Figure 1 A. The authors show iso-G or iso-C nucleotide inhibit the concatemer synthesis.
>Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples

http://www.biomedcentral.com/1471-2164/11/413

Hi everybody

A this time, we have done six ½ Titanium run of cDNA sequencing. We always use SMART (SMARTER kit from Clontech) cDNA preparation without normalization. In the last run, we obtain at least 50% of SMART adaptor concatemer sequence.

Of course, we have followed the Clontech protocol and verified the quality and the amount of our RNA

Is anybody has an hypothesis to explain how this concatemer occur and how to modify the SMART original protocol in order to avoid this problem.

Thanks a lot

[JamesHrastelj]

Repeat insertion with SMARTer Pico library prep

Hi,

I was very interested in the problems you've been having with the Clontech/Takara SMARTer Pico kit. I've just completed a pilot run of RNA-seq using 5 human total RNA samples (input 1-3ng, RINs 5.8-9.7) and have had very strange results.

The cDNA libraries looked good and the sequencing appeared to work well, but the mapping % was highly variable. On looking at the FastQC reports of trimmed reads, the main correlate with mapping % was the prominence of a strange 'saw-tooth' pattern on the per base sequence content (see attached). Could this represent regular insertion of repeat sequences? This effect was independent of RNA quality and input amount.

I read the very helpful aritcle posted by ecPDCNRA, and wondered whether something similar could have occurred with my samples. The odd thing is that there are no over-represented sequences in the bad samples.

If anyone has any advice I'd be extremely grateful!!

Thanks and best wishes!