title: Extraction of ultrashort DNA molecules from herbarium specimens.
abstract: DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.
authors.affiliation: Research Group for Ancient Genomics and Evolution, Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Rafal M
authors.initials: RM
authors.lastname: Gutaker
authors.affiliation: Institute of Archaeological Sciences, University of Tübingen, Tübingen, Germany.
authors.forename: Ella
authors.initials: E
authors.lastname: Reiter
authors.affiliation: Institute of Archaeological Sciences, University of Tübingen, Tübingen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Furtwängler
authors.affiliation: Institute of Archaeological Sciences, University of Tübingen, Tübingen, Germany; Senckenberg Center for Human Evolution and Paleoenvironment, University of Tübingen, Tübingen, Germany.
authors.forename: Verena J
authors.initials: VJ
authors.lastname: Schuenemann
authors.affiliation: Research Group for Ancient Genomics and Evolution, Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Hernán A
authors.initials: HA
authors.lastname: Burbano
mesh_terms: D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D000072441:DNA, Ancient / Q000737:chemistry / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013844:Thiazoles
chemical_list: D002593:Cetrimonium Compounds; D000072441:DNA, Ancient; D018744:DNA, Plant; C101705:N-phenacylthiazolium bromide; D013844:Thiazoles; D000077286:Cetrimonium
keywords: DNA extraction; ancient DNA; herbarium; next-generation sequencing; paleogenomics