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slls9969 03-06-2015 09:01 AM

ion proton. Reads length much shorter than library size?
 
Hi,

Recently I'm doing RNAseq on ion proton. I used ion total-RNA seq v2 kit to make the libraries and checked the size distribution on fragment analyzer. The average size of all libraries was 170-190bp. However, when I sequenced them on ion proton, the average read length is only around 79bp. Does anyone have any suggestions how I can increase the read length? The template kit and sequence kit I'm using is Ion PI Template OT2 200 kit v3 and Ion PI sequencing 200 kit v3. I'm a new user of Ion proton, so any suggestion is appreciated.

RemitoAmigo 03-09-2015 04:57 AM

Hi,

adapters, key sequences, barcodes?

thomasblomquist 03-10-2015 07:44 AM

Ditto with the above. Is this before or after adapter and barcode trimming?

In addition: Most fragment analyzers "phorese" under non-denaturing conditions. Library preparation can lead to heterodimerization between non-homologous captured nucleic acid targets (a lot of complementarity on the ends; adapters/sequencing primers etc). The center portion of these heterodimers bulge out 3-dimensionally and phorese at higher SIZES/Times compared to their True 2-D linear length.

Thus, there will be some discrepancy between the fragment library sizing on electrophoresis, the ladder used and your actual insert size.

-Tom


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