Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing

Abstract

In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced.

Reinstatement of Eschatoporiini Blaisdell, 1906, a unique tribe of blind cavernicolous Tenebrionidae from California, with a new species from Napa County (Coleoptera, Tenebrionidae, Lagriinae)

“…Four gene fragments were amplified: 28S nuclear ribosomal DNA (28S), arginine kinase (ArgK), carbamoyl phosphate synthetase domain of the rudimentary gene (CAD), and wingless ( wg ). These gene fragments were previously sequenced for the Lagriinae sampled in Kanda et al (2015). Polymerase chain reactions (PCRs) were performed on either an Eppendorf Mastercycler ProS or Mastercycler gradient Thermal Cycler using Ex Taq DNA polymerase (TaKaRa) and basic protocols recommended by the manufacturers.…”

“…Polymerase chain reactions (PCRs) were performed on either an Eppendorf Mastercycler ProS or Mastercycler gradient Thermal Cycler using Ex Taq DNA polymerase (TaKaRa) and basic protocols recommended by the manufacturers. Primer pairs and cycler profiles are described in Kanda et al (2015). PCR products were cleaned, quantified, and sequenced at the University of Arizona’s Genomic and Technology Core Facility using a 3730 XL Applied Biosystems automatic sequencer.…”

“…Over the past decade material belonging to a new species of Eschatoporis has been collected from a cave in Napa County, California; thus allowing for a representative of the genus to be sequenced and analyzed within the context of a large pre-existing molecular dataset for the Lagriinae (Kanda et al 2015). This new species, Eschatoporis styx , is described below.…”

Documenting tenebrionid diversity: progress on Blaps Fabricius (Coleoptera, Tenebrionidae, Tenebrioninae, Blaptini) systematics, with the description of five new species

“…The low rate of PCR success results from the fact that we mostly processed specimens collected more than 10 years ago (PCR failed for more than 50 specimens). Because numerous species of interest are distributed in areas that are now hardly accessible, we think that future phylogenetic studies on species of Blaps (especially for the species that are distributed in Northern Africa) will require the intensive use of museomics approaches (see, e.g., Tin et al 2014;Kanda et al 2015).…”

Phylogenetic placement of the Pacific Northwest subterranean endemic diving beetle Stygoporus oregonensis Larson & LaBonte (Dytiscidae, Hydroporinae)

“…PCRs were performed in 25 microliter reactions on either an Eppendorf Mastercycler gradient or Mastercycler ProS using TaKaRa Ex Taq following manufacturer’s protocols. We used primer pairs and amplification conditions described in Miller et al (2013) for 12S, 16S, COI (Pat/Jerry), COII, and H3, and Kanda et al (2015) for wg and the barcoding region of COI (Suppl. material 4).…”