Abi prism 3730 l dna analyzer

The genomic regions surrounding the predicted mutations were amplified using AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, USA). Primers were summarized in Supplementary Table 11.
The PCR products were purified with ExoSAP-IT (Affymetrix, USA) and subjected to Sanger Sequencing (Macrogen, USA). The amplicons containing the predicted genomic mutations were sequenced using BigDye Terminator Cycle Sequencing Kit v3.1 on the ABI Prism 3730×l DNA Analyzer (Applied Biosystems, USA). All Sanger validation figures are in Supplementary Data 1.
To assess the sensitivity of judging absence of a mutation in one phase that is present in the other phase, we studied 15 variants in the panel that are absent in one of the samples in WES, with median WES depth 117 [10-402]. Using CancerScan^{6 (link)}, we found that no read reported the variant in the sample where it was deemed absent by WES (Supplementary Table 12), median CancerScan depth 563 [217-1377].

1419, 1098, 514, 4899, and 2876 base pairs of the rrs, gltA, rOmpA, rOmpB, and sca4 genes respectively was amplified using primers that have been previously described (Fournier et al., 2003 (link)). Sequencing of the amplicons using BigDye v3.1 technology was performed using a GeneAmp PCR System 2400 thermocycler (Applied Biosystems, USA). The product was then analyzed using an ABI Prism 3730×l DNA Analyzer (Applied Biosystems, USA) at the Australian Genomic Research Facility.
Sequencing traces were assembled in the DNASTAR software package (DNASTAR, Inc. USA) and analysed using BLAST analysis software (NCBI) (Altschul et al., 1990 (link)). All sequences have been deposited in GenBank (Table 1). A concatenated tree of the 5 sequences was generated using neighbor-joining and maximum parsimony methods in the MEGA 7 software package.