DOI: 10.5061/dryad.q7m07
title: Data from: Successful recovery of nuclear protein-coding genes from small insects in museums using illumina sequencing

description: 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.
description: CarabidAnalysesFinalMainTreesThe NEXUS file written for Mesquite which was used to test for accurate phylogenetic placement of sequences obtained from Illumina sequencing of carabid museum specimens in the context of a larger phylogeny. The file contains matricies for seven genes and the trees produced by the analyses.BembidionTransversaleGroupAnalysisThe NEXUS file written for Mesquite we used to test for accuracy of Illumina sequences obtained from museum specimen carabids in the context of conspecific and very closely related species. The file contains matricies and phylogenetic trees for four genes.Lagriinae_AnalyzedMatricesThe NEXUS file written for Mesquite which was used to test for the accurate phylogenetic placement of Illumina sequences obtained from museum tenebrionid specimens in a larger phylogeny.FocalGene_QuerySet_TriboliumThe query sequences from the Tribolium castaneum genome which were used to identify fragments of 7 focal genes in reads from Illumina sequenced carabid and tenebrionid museum specimens.NPG_QuerySet_TriboliumQuery sequences from the Tribolium castaneum genome which were used to detect the presence of 67 nuclear protein coding genes in Illumina reads of tenebrionid museum specimens.Bembidion_transversale_DNA3205_67PCGQuery sequences from Bembidion transversale genomic reads which were used to detect the presence of 67 nuclear protein coding genes in Illumina reads of carabid museum specimens.