My project is involved in whole genome sequencing of a few mycobacteria strains. Some mutations were successfully called in some genes and we focus on missense mutation. However, after PCR those regions and sent for Sanger sequencing, the result is negative. The chromatogram shows no mutation in these nucleotides.
Anyone has encountered this problem before?
In the NGS data, was the mutation found in all reads across that region? If not, could there be strain heterogeneity or could you be looking at a minor sequencing artefact. The frequency of the mutant in the NGS data reads would be critical towards the Sanger approach.
The answers to the above might affect the Sanger sequencing. For instance, in the Sanger sequencing, were you directly sequencing the PCR product? or was it cloned first? If it was directly sequenced, you might want to look at the sequence traces and see if there was a hint of the mutant present as a minor form. If you are cloning first, you would need to sequence multiple clones to pick up minor variants.
If both datasets are very clean and disagree, I think you need to consider a large number of possible sample handling problems.
I agree with Prescott, I think you should also check for misalignment in your assembly, i.e. if the reads supportingyour mutation align elsewhere in the genome.
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