Shotgun library preparation and sequencing

To test the integrity of the extracted DNA from the Monarch Oligo extraction protocol, we performed a full NGS analysis, including bioinformatics after sequencing.

Whole genome shotgun libraries were prepared from 10 museum specimens with DNA extracted using the Monarch® PCR & DNA Clean-up Kit, based on a published protocol for degraded DNA samples [25], which was modified in order to incorporate adapter design that enables high sample multiplexing on a single sequencing lane [26–28]. They were sequenced on a 75 bp paired-end Illumina MiSeq run. Percentages of duplicates and adaptors were estimated using FastQC [29]. Removal of low-quality reads and adaptor sequences (Illumina) was performed with BBDuk from BBTools [30].

The origins of these sequences were tested with Kraken2, a taxonomic classification system based on k-mer matches to achieve high accuracy in the classification of sequences. It matches each k-mer within a query sequence to the lowest common ancestor (LCA) of all genomes containing the given k-mer. The k-mer assignments inform the classification algorithm [31]. The standard database of Kraken2 was expanded with a reference genome and mitochondrial genome of the genus Hyles, consisting of assembled long-reads from a single Hyles vespertilio specimen [32].