Hey All,
I have previously done my own library prep for PE100 on HiSeq4000 and my fragment size is around 300bp recommended.
This time when I completed library prep my fragments range from 300-400bp and have failed QC done by the sequencing core.
Some samples have a tiny peak at 113-117bp - the library prep kit I use is NEBnext RNA Ultra Library prep with ribosomal RNA depletion - this kit says that peaks at 80bp and 125bp is an issue and requires clean up.
The sequencing core did a clean up but some samples are still failing. I have written to them to ask exactly what QC parameters are failing.
Bioanalyser traces attached if that helps.
Sample 4-6 were from difficult to handle samples so it isn't a surprise that library prep didn't work.
Sample 23-24 are blank controls.
I guess my main questions are:
1) Have you successfully sequencing PE100 with fragment sizes up to 400bp?
2) Is the small peak at 113-117bp an issue?
Any suggestions / comments are welcome!
Thank you!
I have previously done my own library prep for PE100 on HiSeq4000 and my fragment size is around 300bp recommended.
This time when I completed library prep my fragments range from 300-400bp and have failed QC done by the sequencing core.
Some samples have a tiny peak at 113-117bp - the library prep kit I use is NEBnext RNA Ultra Library prep with ribosomal RNA depletion - this kit says that peaks at 80bp and 125bp is an issue and requires clean up.
The sequencing core did a clean up but some samples are still failing. I have written to them to ask exactly what QC parameters are failing.
Bioanalyser traces attached if that helps.
Sample 4-6 were from difficult to handle samples so it isn't a surprise that library prep didn't work.
Sample 23-24 are blank controls.
I guess my main questions are:
1) Have you successfully sequencing PE100 with fragment sizes up to 400bp?
2) Is the small peak at 113-117bp an issue?
Any suggestions / comments are welcome!
Thank you!
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