High-throughput sequencing of insect specimens with sub-optimal DNA preservation using a practical, plate-based Illumina-compatible Tn5 transposase library preparation method

Abstract

Entomological sampling and storage conditions often prioritise efficiency, practicality and conservation of morphological characteristics, and may therefore be suboptimal for DNA preservation. This practice can impact downstream molecular applications, such as the generation of high-throughput genomic libraries, which often requires substantial DNA input amounts. Here, we use a practical Tn5 transposase tagmentation-based library preparation method optimised for 96-well plates and low yield DNA extracts from insect legs that were stored under sub-optimal conditions for DNA preservation. The samples were kept in field vehicles for extended periods of time, before long-term storage in ethanol in the freezer, or dry at room temperature. By reducing DNA input to 6ng, more samples with sub-optimal DNA yields could be processed. We matched this low DNA input with a 6-fold dilution of a commercially available tagmentation enzyme, significantly reducing library preparation costs. Costs and workload were further suppressed by direct post-amplification pooling of individual libraries. We generated medium coverage (>3-fold) genomes for 88 out of 90 specimens, with an average of approximately 10-fold coverage. While samples stored in ethanol yielded significantly less DNA compared to those which were stored dry, these samples had superior sequencing statistics, with longer sequencing reads and higher rates of endogenous DNA. Furthermore, we find that the efficiency of tagmentation-based library preparation can be improved by a thorough post-amplification bead clean-up which selects against both short and large DNA fragments. By opening opportunities for the use of sub-optimally preserved, low yield DNA extracts, we broaden the scope of whole genome studies of insect specimens. We therefore expect these results and this protocol to be valuable for a range of applications in the field of entomology.

Fig 1. DNA extract concentration measurements (ng) from single bumblebee legs (B. pascuorum and B. lapidarius) stored using 2 different methods.

Specimens were sampled in 2017 from various locations in Norway, Germany and Denmark and stored in ethanol in fieldwork vehicles at ambient summer temperatures for several weeks. Samples were then either stored dry (at room temperature) or in ethanol (at -18°C). See Materials and Methods for details regarding sample collection and DNA extraction. Following extraction, DNA was eluted in 105μL. DNA yields from dry-stored specimens (mean = 298.21±181.03) were significantly greater than from those stored in ethanol (mean = 72.98±51.79; t = 5.45, df = 20.57, P < 0.001).

Fig 2. Fragment size distribution profiles of DNA libraries built from a standardised input of 6ng DNA from each of two B. pascuorum specimens, using different tagmentation reaction times and transposase dilutions.

(a) Libraries were built using four different tagmentation reaction times (5 minutes; 6 minutes; 7 minutes; 8 minutes) with a 1X transposase dilution. (b) Libraries were built using four different transposase dilutions (2X; 4X; 6X; 8X) with a 7 minute tagmentation reaction. RFU (Relative Fluorescence Units) signifies relative amount of DNA. A vertical line indicating a fragment size of 300 bp is included to assist visual comparison of the different treatments. See Materials and Methods for details regarding library preparation protocol.

Fig 3. Comparison of fragment size distribution of 96 pooled bumblebee DNA libraries which were cleaned up with different AMPure XP protocols: A single clean-up with a 0.6 DNA/bead ratio (Pool SP), and a double-sided clean-up using two ratios (0.5 DNA/bead ratio followed by 0.65 DNA/bead ratio) which was performed twice (Pool S4).

RFU (Relative Fluorescence Units) signifies relative amount of DNA.

Fig 4. Sequencing comparisons of two pools of 90 bumblebee libraries.

Bumblebee specimens were sampled between 2017 and 2022 from various locations in Norway, Germany, Denmark and Sweden. Both pools were generated from the same parallel library session. Pool SP was cleaned up with a 0.6 DNA/bead ratio and was sequenced on an Illumina Novaseq SP flowcell. Pool S4 was cleaned up using a double-sided size selection protocol, with a 0.5 DNA/bead ratio clean-up followed by a 0.65 DNA/bead ratio clean-up (twice) and was sequenced on ¼ Illumina Novaseq S4 flowcell. The boxplots compare (a) number of reads; (b) proportion of collapsed reads; (c) endogenous DNA proportion (unique reads only); (d) read length and (e) coverage (fold).

Fig 5. Sequencing comparisons of two combined pools of 90 bumblebee libraries from the same specimens stored using 2 different methods.

Bumblebee specimens were sampled between 2017 and 2022 from various locations in Norway, Germany, Denmark and Sweden and were either stored dry at room temperature (n = 82) or in ethanol at -18°C (n = 8). The box plots compare (a) number of reads; (b) proportion of collapsed reads; (c) endogenous DNA proportion (unique reads only); (d) read length and (e) coverage (fold).