Sample collection and sequencing
Culicoides midges were collected from 17 sites across the United
States and Canada (Table 1). Specimens were collected either as pupae
and reared to adulthood, or as adults using CDC light traps baited with
CO2 and UV light (Bioquip 2836BQ). Individuals
morphologically assigned to the C . variipennis complex
were sorted out from the by-catch and stored in 95% ethanol at -80 °C.
Total DNA was extracted from individuals (females only) using a Puregene
extraction protocol (Gentra Systems, Inc., D-5500A) with the addition of
glycogen (ThermoFisher, R0561) to increase yields. The DNA quality was
checked using gel electrophoresis and DNA concentration was measured
using a Qubit 3.0 fluorometer and a Qubit dsDNA HS assay kit
(Invitrogen, Q33230). A total of 300-400 ng of DNA per sample was sent
to Floragenex, Inc. for library preparation using the protocol from
Truong et al. (2012). DNA was digested using the restriction enzymesMseI and PstI . After PCR amplification, the samples in
each plate were pooled and sequenced on a lane of single-end 100bp
sequencing on a HiSeq4000 at the University of Oregon Genomics Facility,
Eugene, OR.