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Old 08-25-2017, 07:44 AM   #1
cystron
Junior Member
 
Location: Texas

Join Date: Jan 2011
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Default Where do NGS projects most often Fail?

When thinking about a new project, where do you see the most failures?
1. DNA/RNA extraction
2. Library Prep
3. Sequencing Run
4. Alignment / Assembly
5. Secondary/Tertiary analysis and BioInformatics

At what point do you finally feel like a project is "safe"?
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Old 08-25-2017, 09:09 AM   #2
GenoMax
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I am not sure if this question has a generally applicable answer. Projects can fail at any step mentioned in your list. A project can never be "safe" (to borrow your phrase) until the data is analyzed and the paper written :-)
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Old 08-25-2017, 04:09 PM   #3
gringer
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Experimental design: not choosing appropriate controls, too few biological replicates, or doing sequencing on the wrong cell population (e.g. whole-blood RNASeq for an obesity study).
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Old 08-28-2017, 06:29 AM   #4
Genetic Librarian
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Quote:
Originally Posted by gringer View Post
Experimental design: not choosing appropriate controls, too few biological replicates, or doing sequencing on the wrong cell population (e.g. whole-blood RNASeq for an obesity study).
This!

Biggest mistake is too few replicates. Individual samples can drop out for a variety of reasons, so easily your glorious n=3 study has treatments with only 2 replicates in them. And you can not easily repeat the missing samples without having a potential batch effect confounder.

Also, from my experience as a core lab head, usually it is "**** in, **** out", meaning most library preps / sequencing runs fail because input material was not ideal (but customer wanted to try anyway after being contacted following QC).
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