Dear All,
I've recently started performing RNA-seq experiments to sequence whole viral genomes. I started with using the Script-Seq kit (originally sold by epicenter). I start the protocol with total RNA (required according to manual: mRNA or ribosomal depleted RNA) but limited to the input concentration.
I follow the protocol as suggested with a slight modification. I use 0.85X AmpureXP beads after PCR enrichment instead of 1X.
The quality of the run was quite bad. Clustering was on the upper limit for the V3 chemistry (range 1200-1400 k/mm2) with 86% passing filter. I was surprised to see the Qscore! Is it because there is a big portion of the fragments shorter than the read length?
Has anyone modified this protocol in such a way to make it more feasible on a 300x2 bp run? Is there any other step in the protocol that might lead to those short fragments?
I would appreciate the help with this! I attached an agilent trace for the libraries used (#1,2,11 did not work) and some screenshots from the analysis software.
Thank you!
Exo
I've recently started performing RNA-seq experiments to sequence whole viral genomes. I started with using the Script-Seq kit (originally sold by epicenter). I start the protocol with total RNA (required according to manual: mRNA or ribosomal depleted RNA) but limited to the input concentration.
I follow the protocol as suggested with a slight modification. I use 0.85X AmpureXP beads after PCR enrichment instead of 1X.
The quality of the run was quite bad. Clustering was on the upper limit for the V3 chemistry (range 1200-1400 k/mm2) with 86% passing filter. I was surprised to see the Qscore! Is it because there is a big portion of the fragments shorter than the read length?
Has anyone modified this protocol in such a way to make it more feasible on a 300x2 bp run? Is there any other step in the protocol that might lead to those short fragments?
I would appreciate the help with this! I attached an agilent trace for the libraries used (#1,2,11 did not work) and some screenshots from the analysis software.
Thank you!
Exo
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