Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA

Abstract

Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10 ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens.

Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods

“…Krehenwinkel and Pekar () suggest that smaller specimens are likely more quickly preserved than larger specimens, which may lead to good DNA recovery. Our results may also reflect this finding, and are congruent with other studies that have shown successful NGS success of museum specimens of small insects (Sproul & Maddison, ), as well as those utilizing ddRAD to successfully sequence traditionally collected and curated bees (Vaudo et al, ). Vaudo et al () specifically found that specimen age did not affect number of polymorphic loci or coverage depth, and similarly showed that collection method had the most significant effects on overall ddRAD assembly quality.…”

“…One past ddRAD study using whitefish specimens found that proportions of low‐quality reads increased exponentially for specimens with high levels of degradation (extracted 96 hr post‐euthanasia), while specimens with moderate degradation (12–48 hr post‐euthanasia) produced similar results to the non‐degraded DNA, including high levels of depth and numbers of polymorphic loci (Graham et al, ). Other studies have shown that degraded DNA from museum specimens (collected 50–100 years ago) can be successfully used for RAD procedures (Haponski, Lee, & Foighil,; Sproul & Maddison, ; Tin, Economo, & Mikheyev, ), but these studies have not examined the impacts of differing sampling techniques (e.g. netting vs. trapping), different DNA extraction methods, or use of different focal species on ddRAD sequencing success.…”

Optimized DNA extraction and library preparation for minute arthropods: Application to target enrichment in chalcid wasps used for biocontrol

“…When DNA quantities are too low, an extra WGA step can be performed. We definitely agree with Sproul and Maddison () who emphasize how important it is not to waste DNA obtained from irreplaceable specimens (whether fresh or historical). It is even more important to capitalize on existing collections as collecting samples for large‐scale studies may be more and more difficult, given that many countries have imposed restrictive access regulations, even to academic researchers, to reduce the risk of supposed biopiracy (Divakaran Prathapan, Pethiyagoda, Bawa, Raven, & Rajan, ).…”

“…To our knowledge, this study is the second after Sproul and Maddison () to demonstrate success in library preparation from such low input using commercial kits, and the first to report successful sequencing of >1,000 low copy genes in 96 specimens in parallel, from such low input and processing time. Our optimizations differ from what was proposed by Sproul and Maddison (). First, we tried to optimize DNA extraction itself by using overnight lysis with gentle mixing to preserve fragile specimens, heated elution buffer and increased incubation time before elution.…”

Sequence capture phylogenomics of historical ethanol‐preserved museum specimens: Unlocking the rest of the vault

“…Previous UCE studies demonstrated recovery of complete mitochondrial genomes (do Amaral et al, ; Zarza et al, ), and successful combination of UCE‐derived COI sequences with traditional Sanger‐sequenced COI data (Derkarabetian et al, ; Hedin, Derkarabetian, Blair, et al, ). Although not sequence capture studies, the retrieval of many traditional loci from museum samples has been demonstrated previously using modern sequencing technologies (Prosser et al, ), including the nuclear ribosomal complex (Sproul & Maddison, ). As in Branstetter, Longino, Ward, & Faircloth (), our study extends the utility of sequence capture of UCEs to include the ribosomal complex, here 18S and 28S rRNA, from both fresh and museum samples, furthering the ability to combine sequence capture data with many traditional Sanger‐sequenced datasets.…”