Bigdye version 3

A total of four ORF5 PCR amplified products were sequenced (Beckman Coulter Genomics) using Applied Biosystems BigDye version 3.1. The sequences were assembled using software Ridom TraceEditPro 1.2.2., then compared to other EAV sequences using a BLAST web-based program (http://www.ncbi.nlm.nih.gov/BLAST). Nucleotide sequences were aligned in keeping with the Clustal W method. The phylogenetic trees were constructed using the Maximum Likelihood method and the tree’s statistical robustness was assessed by bootstrap resampling (1000 datasets) of the multiple alignments. Phylogenetic reconstruction was carried out using MEGA software version 5.1. [18 (link)]. The sequence described in the article was submitted to GenBank and registered under accession number KX 645659.

DTU identification was confirmed by sequencing PCR amplicons from the miniexon intergenic region after purification using a PureLinkTM Quick PCR purification kit (Invitrogen®). For samples that showed more than one amplification band for a molecular marker, each band was excised and recovered using ZymocleanTM Gel DNA Recovery Kit (Genesee Scientific®) according to the manufacturer’s instructions. All samples were sequenced by GENEWIZ, Inc (South Plainfield, NJ) using Applied Biosystems’ BigDye version 3.1 and Applied Biosystems’ 3730xl DNA Analyzer. The sequences were edited manually using MEGA version 5 software [28 (link)]. A total of 15 sequences of sufficient quality were obtained for analysis (S26MS NOLA, S18MH NOLA, S26ML NOLA, S38MH2 NOLA, S14MH NOLA, S52RH2 NOLA, S38MH1 NOLA, S52RH1 NOLA, S6MM NOLA, S6ML NOLA, S7MH NOLA, S9MS NOLA, S29MH NOLA, S29ML NOLA, and S26MHNOLA (Accession numbers: KM376435- KM376449)).

PCR products from nested-PCR and qPCR with SYBR green were sequenced bidirectionally using the same primers and BigDye version 3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. After purification, the products were sequenced on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The sequences obtained were then compared with those of other flaviviruses, alphaviruses, and leptospiral sequences using the BLASTn program (available online: blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 8 October 2021).

We first aimed to determine whether the Ae. albopictus population in Mecklenburg County had any genetic mutations that would indicate resistance to pyrethroid insecticides. We therefore destructively extracted DNA from whole mosquitoes for use in polymerase chain reaction (PCR) using Qiagen DNeasy Isolation Kits (Qiagen Sciences, Germantown, MD, USA). For all samples, we amplified and sequenced two regions of kdr (domain II, 381 bp; domain IV, 280 bp) using the AegSCF20/AegSCR21 and AlbSCF6/AlbSCR8 primer pairs, respectively (Appendix A). Amplification of kdr domain III was unsuccessful. The thermocycler conditions were identical for kdr domains II and IV, an initial denaturing step at 96 °C for 10 min, 40 cycles of 30 s at 96 °C, 30 s at 55 °C, and 45 s at 72 °C, with a final extension step of 10 min at 73 °C (Appendix A, Table A1). All mosquito PCR products were cleaned using exonuclease I and shrimp alkaline phosphatase (Fisher Scientific, Pittsburgh, PA, USA). Primer extension sequencing was performed by Genewiz (South Plainfield, NJ, USA) using Applied Biosystems BigDye version 3.1. The reactions were then run on Applied Biosystem’s 3730xl DNA Analyzer.
We used MegaX [31 (link)] and BioEdit [32 ] to assemble and form contigs of our forward and reverse reads.

PCR amplification of LZTR1 was performed as described in Table S1. An additional amplicon was included to capture intron 16 where variants can lead to retention of an alternative exon.15 Following enzymatic purification, Sanger sequencing was performed using BigDye (version 3.1) and the ABI 3730XL (Applied Biosystems, Foster City, California).
Where parental samples were unavailable, compound‐heterozygous variants were phased by allele‐specific PCR whereby the 3′‐base of the first primer was complementary to either the wild‐type or mutant allele (Table S1). Sanger sequencing was then performed to determine the sequence at the second locus. A similar method was used to phase a de novo variant where the closest informative SNP was too distant to be phased by Illumina read‐pairs (Table S1).
For one family where the patient underwent exome sequencing as a singleton, maternity/paternity were confirmed by genotyping nine short tandem repeat (STR) loci using the AuthentiFiler PCR Amplification Kit (ThermoFisher Scientific, Waltham, Massachusetts).