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Old 03-06-2015, 09:01 AM   #1
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Location: California

Join Date: Mar 2015
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Default ion proton. Reads length much shorter than library size?


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.
slls9969 is offline   Reply With Quote
Old 03-09-2015, 04:57 AM   #2
Location: Ottawa, Canada

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adapters, key sequences, barcodes?
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Old 03-10-2015, 07:44 AM   #3
Location: Ohio

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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.

thomasblomquist is offline   Reply With Quote

ion proton, read length

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