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FastQC

Below is a detailed description of what to look for in a FastQC report of a bisulfite-sequencing experiment.

Quality control metrics

All metrics are important, but some are more relevant for bisulfite-sequencing than others. The most important metrics are:

  • Per base sequence quality
  • Sequence length distribution
  • Sequence duplication levels
  • Adapter content

Note: You will get an error with "Per base sequence content" if your bisulfite conversion step was done correctly. FastQC expects an approximately equal distribution between AT/GC, but bisulfite conversion explicitly converts unmethylated C -> T, leaving only methylated C detected as C by sequencing. If you have paired-end sequencing, one mate (R1, for example) will have a much higher percentage of T and lower percentage of C, and the other mate (R2) will have a much higher percentage of A and lower percentage of G.