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Biotechniques. 2015 Dec 1;59(6):368-71. doi: 10.2144/000114364. eCollection 2015 Dec.

New insights on single-stranded versus double-stranded DNA library preparation for ancient DNA.

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Center for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
Center for Biological Sequence Analysis, Technical University of Denmark, Kgs. Lyngby, Denmark.
Trace and Environmental DNA Laboratory, Department of Environment and Agriculture, Curtin University, Perth, Australia.


An innovative single-stranded DNA (ssDNA) library preparation method has sparked great interest among ancient DNA (aDNA) researchers, especially after reports of endogenous DNA content increases >20-fold in some samples. To investigate the behavior of this method, we generated ssDNA and conventional double-stranded DNA (dsDNA) libraries from 23 ancient and historic plant and animal specimens. We found ssDNA library preparation substantially increased endogenous content when dsDNA libraries contained <3% endogenous DNA, but this enrichment is less pronounced when dsDNA preparations successfully recover short endogenous DNA fragments (mean size < 70 bp). Our findings can help researchers determine when to utilize the time- and resource-intensive ssDNA library preparation method.


DNA library preparation; ancient DNA; next-generation sequencing; paleogenomics

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