Bigdye terminator cycle sequencing kit version 1

Genomic DNA was extracted from peripheral blood leukocytes according to established protocols. Genotyping of SNPs was carried out by direct sequencing, TaqMan SNP genotyping (Applied Biosystems, Foster City, USA) or by primer extension of multiplex PCR products and subsequent allele detection by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF; Sequenom, San Diego, USA). Direct sequencing was performed with the Big Dye Terminator Cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, U.S.A.) according to the manufacturer’s instructions. Reactions were analyzed with an ABI Prism 3130xl sequencer (Applied Biosystems). TaqMan pre-designed SNP genotyping assays (Applied Biosystems) were used according to the manufacturer’s instructions. The rs144087548 variant was genotyped by polymerase chain reaction (forward primer: 5′-CGC AGA CAT GAT GCT GGG GGT-3′; reverse primer: 5′-ACA TGC AAG ACG GGG AAT TGA-3′) followed by HpyCH4III digestion (New England Biolabs, Ipswich, USA) and restriction fragment length analysis. All SNPs showed high genotyping quality with an average call rate >98 % in each of the five case–control samples.

PCR amplification of the different 27 CFTR exons and the intron-exon boundaries were initially performed on the patients extracted nucleic acid as previously reported by Chou et al [13 (link)]. Amplified PCR products were sequenced bi-directionally using BigDye^® Terminator Cycle Sequencing kit version 1.1 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA) according to the manufacturer recommendations. Excess primers and dye terminators were then removed by centrifugation through a Centrisep spin column containing sephadex G-50 resin (GE Health Life Science, Pittsburgh, USA). Purified sequencing reaction products were then loaded up to Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA).

DNA from termites and tissues was screened for the presence of Wolbachia via Polymerase Chain Reaction (PCR) using wsp primers: wsp81F (5’-TGG TCC AAT AAG TGA TGA AGA AAC-3’) and wsp691R (5’-AAA AAT TAA ACG CTA CTC CA-3’) [42 (link)]. Each PCR reaction was performed in a total volume of 12.5μL, composed of 2X Taq PCR Master Mix (Qiagen, France), 0.5μM of each primer, 2.35mM MgCl2, 0.016mg/mL Bovin Serum Albumine and 2μL of template DNA. The conditions for amplification were initial denaturation at 95°C for 3 minutes then 35 cycles of (1) denaturation at 95°C for 30 seconds, (2) annealing at 45°C for 30 seconds, (3) extension at 72°C for 1.30 minutes and a final extension at 72°C for 10 minutes. A negative (sterile water) and a positive control (known infected queen DNA) were included in each PCR run. Each sample was tested twice and was considered to be infected when at least one of the PCRs was positive. Sequencing of wsp gene was realized for up to three sequences per colony and for salivary glands using BigDye Terminator Cycle Sequencing kit version 1.1 (Applied Biosystems). Sequences were obtained using an automatic DNA sequencer (AppliedBiosystems, ABI PRISM 310). Accession numbers are listed in Table 1.

A fragment of the cytochrome c oxidase subunit I mitochondrial gene (COI) was amplified and sequenced using the universal primers LCO1490 and HCO2198 [34] . For the I20 and IR populations, sequences were taken from Torres-Leguizamon et al. [27] (GenBank accession numbers JN381881–JN381930). Each amplification mixture (25 µl) contained 10 ng DNA, 12.5 µl of Taq PCR Master Mix (Qiagen, Hilden, Germany) and 0.25 µM of each primer. Polymerase chain reactions were performed using an initial denaturation step at 94°C for 3 min, followed by 40 cycles of the three following steps: denaturation at 94°C for 30 s, annealing at 49°C for 1 min and extension at 72°C for 1 min 30 s. The final extension was done at 72°C for 10 min. PCR products were purified using Microclean (Microzone Limited, Haywards Heath, UK). Both strands of amplicons were sequenced using Big-Dye Terminator Cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, CA, USA) according to the standard protocol used in the genomic platform at the Mondor Institute of Biomedical Research (Créteil, France). Sequences were deposited in GenBank (Accession numbers: KF856627–KF856710).

We used previously reported KRAS mutation data in the same cohort (29 (link)). In brief, genomic DNA was extracted from the FFPE tissue blocks by a QIAmp DNA Mini Kit (Qiagen, Valencia, CA, USA) as previously described (29 (link)). The KRAS genes were amplified using the following primers: forward, 5′-TGACATGTTCTAATATAGTCAC-3′, and reverse, 5′-ACAAGATTTACCTCTATTGTT-3′). The PCR reaction volume was 25 μl, including 0.3 μM of each primer and AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturing at 95°C for 50 s, annealing for 50 s, elongation at 72°C for 1 min, and a final elongation at 72°C for 7 min. The PCR amplicons were purified by a QIAquick PCR Purification Kit (Qiagen) and sequencing reactions were performed in the forward and reverse directions using the BigDye Terminator Cycle Sequencing Kit, version 1.1 (Applied Biosystems).