Balancing Barcoding and Genomics: gDNA Quality in Insect Vouchers after HotSHOT DNA extraction

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Abstract DNA extraction with an alkaline buffer system called “HotSHOT” is widely used for barcoding because it is rapid, inexpensive, and voucher preserving, but it remains unclear whether sufficient genomic DNA (gDNA) remains in small vouchers for downstream use in genomics. We here evaluate gDNA quality and quantity before and after HotSHOT treatment of 11 insect families representing six orders. Some specimens were flash frozen immediately after collection, while others were kept for one week at tropical temperatures in ethanol to mimic Malaise trap conditions. Encouragingly, we show that gDNA of sufficiently high quality and quantity for genomic sequencing remained in specimens treated with HotSHOT. We also show that DNA integrity was strongly influenced by field storage with specimens exposed to Malaise trap conditions showing such pronounced degradation that the standard HotSHOT treatment no longer significantly altered DNA quality. For control material, HotSHOT treatments involving longer exposure to high temperature led to smaller fragment lengths with the effect apparently being influenced by the degree of specimen sclerotization. Our results thus suggest that optimized HotSHOT treatments, together with carefully controlled pre-extraction storage, preserve voucher gDNA of sufficient quality for downstream genomic analyses with both short-read and possibly even some long-read sequencing technologies. Our protocol selection guidelines improve voucher gDNA preservation in HotSHOT-treated samples. This is particularly important for many species which are only known from one or few specimens discovered during barcoding projects.

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