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Old 03-09-2018, 02:26 AM   #5
JamesHrastelj
Junior Member
 
Location: Cardiff, Wales, UK

Join Date: Mar 2018
Posts: 3
Default 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!
Attached Files
File Type: pdf SMARTerPilot_Read1.pdf (373.5 KB, 19 views)

Last edited by JamesHrastelj; 03-09-2018 at 04:08 AM.
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