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

Nathan Wales*, Christian Carøe, Marcela Sandoval-Velasco, Cristina Gamba, Ross Barnett, José Alfredo Samaniego, Jazmín Ramos Madrigal, Ludovic Orlando, M. Thomas P. Gilbert

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Article number114364
Pages (from-to)368-371
Number of pages4
Issue number6
Publication statusPublished - 1 Dec 2015


  • Ancient DNA
  • DNA library preparation
  • Next-generation sequencing
  • Paleogenomics

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