In addition to sources in the wet lab, bias can be introduced by any of the computational steps in the sequencing pipeline. Signal-processing and base calling limitations could result in under-representation or increased error rates in some locations, as can inaccurate alignment. An inaccurate reference or sample-reference differences can cause coverage or accuracy variations that may be misdiagnosed as sequencing bias. Therefore, detecting bias is only the first step and must be followed by more detailed experiments to assign responsibility to the library preparation, sequencing, or computational stages.
