Cost-efficient high throughput capture of museum arthropod specimen DNA using PCR-generated baits

Abstract

131. Gathering genetic data for rare species is one of the biggest remaining obstacles in 14 modern phylogenetics, particularly for megadiverse groups such as arthropods. Next 15 generation sequencing techniques allow for sequencing of short DNA fragments 16 contained in preserved specimens >20 years old, but approaches such as whole genome 17 sequencing are often too expensive for projects including many taxa. Several methods of 18 reduced representation sequencing have been proposed that lower the cost of sequencing 19 per specimen, but many remain costly because they involve synthesizing nucleotide 20 probes and target hundreds of loci. These datasets are also frequently unique for each 21 project and thus generally incompatible with other similar datasets. 22 2. Here, we explore utilization of in-house generated DNA baits to capture commonly 23 utilized mitochondrial and ribosomal DNA loci from insect museum specimens of various 24 age and preservation types without the a priori need to know the sequence of the target 25 loci. Both within species and cross-species capture are explored, on preserved specimens 26 ranging in age from one to 54 years old. 27 3. We found most samples produced sufficient amounts of data to assemble the nuclear 28 ribosomal rRNA genes and near complete mitochondrial genomes and produce well-29 resolved phylogenies in line with expected results. The dataset obtained can be 30 straightforwardly combined with the large cache of existing Sanger-sequencing-generated 31 data built up over the past 30 years and targeted loci can be easily modified to those 32 commonly used in different taxa. Furthermore, the protocol we describe allows for 33 inexpensive data generation (as low as ~$35/sample), of at least 20 kilobases per 34 3 specimen, for specimens at least as old as ~1965, and can be easily conducted in most 35 laboratories. 36 4. If widely applied, this technique will accelerate the accurate resolution of the Tree of Life 37 especially on non-model organisms with limited existing genomic resources.38

Hybrid enrichment of poorly preserved museum specimens refines homology hypotheses in a group of minute litter bugs (Hemiptera: Dipsocoromorpha: Schizopteridae)

“…Libraries were prepared without shearing, otherwise following Knyshov et al . (). Hybrid captures followed a pooled design from Knyshov et al .…”

“…The PCR‐generated baits for ribosomal and mitochondrial genes were prepared as in Knyshov et al . () using the same 5′ blocked primers. To reduce costs, we relied on long‐range PCR to amplify the ribosomal operon and mitochondrion in the lowest possible number of reactions.…”

“…Hybrid captures followed a pooled design from Knyshov et al . (), where individual libraries were pooled by eight to 12 per capture reaction. Enriched pools were mixed with bait amplicons and two unenriched benchmarking libraries (for samples scpp7 and scpp54) and sequenced on c .…”

“…We therefore aimed to advance cost‐efficient ways to sample molecular data from poorly preserved museum specimens for large integrative taxonomic projects. For this reason, we modified and tested on insects (Knyshov et al ., ) a previously developed hybrid capture approach (Maricic et al ., ). The approach targets only selected commonly utilized loci (e.g.…”

“…Specifically, we aimed to: (i) use hybrid enrichment approaches to obtain sequence data for low‐quality samples not accessible to traditional Sanger sequencing and sample c . 50% of the estimated species‐level diversity; (ii) push the performance limits of our model study (Knyshov et al ., ) by including specimens of older age, with lower amount of extracted DNA, and visibly contaminated by fungi; and (iii) use ASR of several putatively genus‐diagnostic features, including MSOs, to test primary homology hypotheses and shape the genus‐level classification of the group for an upcoming taxonomic revision.…”