DNA extracted from herbarium specimens is highly fragmented and decays at a faster rate than DNA from ancient bones. Therefore, it is crucial to utilize extraction protocols that retrieve short DNA molecules. Improvements in extraction and library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. We adapted those improvements to extraction protocols for herbarium specimens and evaluated their performance by shotgun sequencing, which allows an accurate estimation of the distribution of fragment lengths. Extraction with PTB buffer decreased median fragment length by 35% when compared to 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 when we used single-stranded instead of double-stranded library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens and will help to unlock the genetic information stored in herbaria.
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