Question: Consequences of degraded DNA for Sequencing
3
gravatar for rmf
4.6 years ago by
rmf1.0k
rmf1.0k wrote:

I have some moderately degraded DNA (DIN < 6) which will be used for standard library prep followed by illumina paired end sequencing. I was wondering what would be the consequences of degraded material with regard to downstream analyses such as SNP calling, structural variant detection etc.

I have been working with RNA preps where degraded mRNA would cause a 5' bias in resulting library when using polyA capture approach. So degraded RNA matters. Is there any such effects on degraded DNA? Long strand DNA will have to sheared anyway during library prep to some shorter insert sizes. So does it even matter that I start with degraded DNA?

Link to DIN http://www.agilent.com/cs/library/applications/5991-5258EN.pdf

dna sequencing ngs • 2.5k views
ADD COMMENTlink modified 4.6 years ago • written 4.6 years ago by rmf1.0k

Have you checked the sizes of your DNA template, e.g. on gel or bio-analyzer or alternatives? The impact is probably not as much a problem as in RNA-seq (unless you want long reads or long mate pairs)

ADD REPLYlink written 4.6 years ago by WouterDeCoster45k
1

I can agree for those wanting to do long reads like Pacbio or something, highest DIN scores with minimal degradation would be preferred. But that shouldn't be a problem for 125 or 150 bp Illumina sequencing.

ADD REPLYlink modified 4.6 years ago • written 4.6 years ago by rmf1.0k

Exactly. In addition, there are repair kits to (partially?) take care of nicked and damaged DNA, e.g. https://www.neb.com/products/m6630-nebnext-ffpe-dna-repair-mix

ADD REPLYlink written 4.6 years ago by WouterDeCoster45k

Perhaps you should look at how people are handling/analysing other degraded DNA samples (FFPE/ancient DNA workflows)

ADD REPLYlink written 4.6 years ago by User 5913k
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