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7.8 years ago
firestar
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1.6k
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
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)
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.
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
Perhaps you should look at how people are handling/analysing other degraded DNA samples (FFPE/ancient DNA workflows)