A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E-4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible.