Bigdye terminator v1.1 kit

For sequencing PCR amplification products resulting in multiple bands, the PCR products were cloned by TA cloning (TA Cloning Kit; Thermo Fisher Scientific) following the manufacturer’s protocol. Plasmid constructs were verified by Sanger sequencing using the sequencing primers listed in Table S2. Plasmids were extracted from bacterial cultures using silica-based purification columns (Monarch Plasmid Miniprep Kit; New England Biolabs) and sequenced using the BigDye Terminator v.1.1 kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The same sequencing protocol was used to verify the success of gRNA cloning in the different backbone vectors. Sequencing of PCR amplicons resulting in a single band was carried out using the BigDye Terminator v.1.1 kit according to the manufacturer’s protocol. Sequencing reactions were resolved on an ABI PRISM 3130xl Genetic Analyzer.

Viruses were passaged on confluent Vero E6 cells infected at a MOI of 0.01 to follow the emergence of compensatory mutations over five sequential rounds of infections. RNA was extracted from infected cells at each passage using TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. 3′-rapid amplification of cDNA ends (3′ RACE) was then carried out using a poly(A) polymerase tailing kit (Epicentre Biotechnologies, Tebu-Bio, Le Perray en Yvelines, France). Briefly five micrograms of total RNA was polyadenylated in a 20-μL reaction mixture for 7 min at 37 °C. The RNA was then purified, and reverse transcription was carried out using the oligo(dT) 3′ RACE-adaptor primer (AP) (Invitrogen) or primers specific for the M segment and avian myeloblastosis virus reverse transcriptase (RT) (Promega, Charbonnières, France). This step was followed by polymerase chain reaction (PCR) using the KOD polymerase (Toyobo, Merk, Darmstadt, Germany) and specific primers and/or 3′ RACE-AP. The resulting RT-PCR products were separated by agarose electrophoresis, and the DNA bands with the correct sizes were purified using the QIAquick purification kit (Qiagen, Courtaboeuf, France) and then sequenced according to standard protocols using the BigDye terminator v1.1 kit (Applied Biosystems, Villebon-sur-Yvette, France).

Before sequencing, a PCR sequencing reaction for the PCR products of EGFR exons 18, 19 and 21 was performed using a BigDye Terminator v1.1 kit (Applied Biosystems, Foster City, Calif., USA) according to the kit instructions. All PCR assays were carried out in a volume of 20 µl that included 1 µl of purified PCR products, 8 µl of BigDye (2.5×) and 1 µΜ of primers (3.2 pmol/µl). The PCR sequencing reaction was performed at 96°C for 1 min, and then 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. Sequencing was carried out in an ABI 3730 genetic analyzer (Applied Biosystems). All sequence variants were confirmed by sequencing the products of independent PCR amplifications.

EGFR and KRAS mutation data of ALK-negative and ALK-positive groups were investigated. The mutation analyses of EGFR (exon 18, 19, 20, and 21) and KRAS (exons 2 and 3) were examined using direct sequencing-polymerase chain reaction (PCR). Genomic DNA was extracted from the FFPE tissue and purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany). Bidirectional sequencing was performed using the BigDye Terminator v 1.1 kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130xl genetic analyzer (Applied Biosystems).

Mutational analysis of KRAS exon 2 was performed by direct Sanger sequencing of PCR products amplified from genomic DNA. PCR was performed in a 20-µL volume containing 100 ng of template DNA, 10× PCR buffer, 0.25 mM dNTPs, 10 pmol primers, and 1.25 U Taq DNA polymerase (iNtRON, Daejeon, Korea). PCR products were electrophoresed on 2% agarose gels and purified with a QIAquick PCR purification kit (Qiagen). Bidirectional sequencing was performed using the BigDye Terminator v1.1 kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130xl genetic analyzer (Applied Biosystems). Chromatograms were manually reviewed for sequence analysis. Confirmatory re-sequencing from replicate PCR amplification reactions was performed for any sequences that were ambiguous. The results were marked as mutation positive if a mutation was detected in both the forward and reverse DNA strand.