E gel ibase real time transilluminator

PCR products were deemed worthy of sequencing when producing a strong, clear band at the correct product size when visualised using an Invitrogen E-Gel iBase Real-Time Transilluminator with 2% SYBR safe E-Gel EX agarose gels run for 10 mins. Products were sent to Source BioScience (Nottingham, UK) for cleanup prior to forward and reverse sanger sequencing. The MLST primers used were gene-specific and in the case of MLST genes just the M13 primers (M13_adaptor_F: 5’-TGTAAAACGACGGCCAGT-3’ and M13_adaptor_R: 5’-CAGGAAACAGCTATGACC-3’) were used if these adaptors were included in the initial PCR to generate the product. MEGAX
^{47 (link)}
was used for all analysis of sequences with manual checking of both forward and reverse chromatograms. Editing of sequences included trimming and then alignment to produce consensus sequences was undertaken using ClustalW. Nucleotide BLAST (NCBI) database queries and searches against the
Wolbachia MLST database were combined to determine if new alleles and strain types were present in our collection. We also submitted our sequences to GenBank and obtained accession numbers.

PCR products were separated and visualised using 2% E-gel EX agarose gels (Invitrogen) with SYBR safe and an Invitrogen E-gel iBase Real-Time Transilluminator. PCR products were submitted to Source BioScience (Source BioScience Plc, Nottingham, UK) for PCR reaction clean-up, followed by Sanger sequencing to generate both forward and reverse reads. Sequencing analysis was carried out in MEGA7^{82 (link)} as follows. Both chromatograms (forward and reverse traces) from each sample was manually checked, analysed, and edited as required, followed by alignment by ClustalW and checking to produce consensus sequences. Consensus sequences were used to perform nucleotide BLAST (NCBI) database queries, and Wolbachia gene loci sequences were used for searches against the Wolbachia MLST database (http://pubmlst.org/wolbachia)⁸³ . If a sequence produced an exact match in the MLST database, we assigned the appropriate allele number.

Mosquitoes were morphologically identified as Anopheles gambiae sensu lato (s.l.) in the field prior to CDC bottle bioassay testing. A sub-set of 480 samples were further identified using an end point PCR assay developed by Santolamazza et al. [12 (link)]. This assay amplifies the SINE200 insertion, a highly repetitive ~ 200 bp element which is widespread in the An. gambiae sensu stricto (s.s.) genome [12 (link)]. Samples were prepared with forward (5′-TCGCCTTAGACCTTGCGTTA-3′) and reverse (5′-CGCTTCAAGAATTCGAGATAC-3′) primers, and amplifications performed in 20 µL reactions containing 2 µL cDNA, 2 µL of each primer (10 µM), 4 µL H₂O, and 10 µL 2× Hot Start Taq PCR Master Mix (New England Biolabs). The cycling conditions in the Bio-Rad T100™ Thermal Cycler were: 10 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 54 °C, and 60 s at 72 °C; then 10 min at 72 °C. Products were run on 2% agarose gels in an Invitrogen E-gel iBase™ Real-Time Transilluminator. Amplification products of 479 bp or 249 bp were considered indicative of Anopheles coluzzii or An. gambiae s.s., respectively; both bands indicate a hybrid individual. The An. gambiae s.s. form has an identical banding pattern to that of Anopheles melas and Anopheles quadriannulatus (249 bp), however, due to the geographical location of sampling, it is highly unlikely that these species were present.