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Old 09-02-2016, 03:59 PM   #1
lilichen
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Location: LOS ANGELES

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Unhappy good QC mRNA seq samples with super low alignement

Hi, Everybody:

We just completed mRNA seq for some human cells bulk samples.
The QC for those seq data is good. But when we run the alignment for those samples, super low alignment rate come out ( ~50%). We built those lib samples by the pico-input stranded mRNA prep kit from Clontech.

Is there any body have experience for this wired low alignment rate issue?
Is there something wrong about the quality of original samples? or lib prep process? or seq running process?

Any advice would be appreciated.
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Old 09-03-2016, 07:17 AM   #2
GenoMax
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Take a sample of the reads that did not align and check using NCBI blast. Did you use an aligner that is splice aware and use appropriate settings for it?
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Old 09-03-2016, 04:02 PM   #3
lilichen
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Quote:
Originally Posted by GenoMax View Post
Take a sample of the reads that did not align and check using NCBI blast. Did you use an aligner that is splice aware and use appropriate settings for it?
Hi, GenoMax: Thanks for your advices. The specialist in the Bioinformatics service helped us with the data analysis. So the setting for those data analysis should be fine. The software used for seq data analysis is Partek flow. That worked good for our last programs.
I would take samples to check using NCBI blast like you adviced.
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Old 09-04-2016, 04:22 PM   #4
nucacidhunter
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Quote:
Originally Posted by lilichen View Post
Hi, Everybody:

We just completed mRNA seq for some human cells bulk samples.
The QC for those seq data is good. But when we run the alignment for those samples, super low alignment rate come out ( ~50%). We built those lib samples by the pico-input stranded mRNA prep kit from Clontech.

Is there any body have experience for this wired low alignment rate issue?
Is there something wrong about the quality of original samples? or lib prep process? or seq running process?

Any advice would be appreciated.
Alignment rate to genome should be over 80% for libraries prepared with this kit.
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