Table 1.

Sources of main bias in RNA-seq.

Bias sources
Sample preservation (1) Degradation of RNA: such as tissue autolysis; nucleic acid degradation and cross-linking during the preparation of formalin-fixed; formalin-fixed paraffin-embedded (FFPE) [6] (2) RNA extraction: such as using TRIzol [12] (3) Alien sequence contamination [73] (4) Low-quality and/or low-quantity RNA [23]
Library preparation (1) mRNA enrichment bias: such as 3′-end capture bias [74] (2) RNA fragmentation bias [31] (3) Primer bias: such as random hexamer bias; mispriming; nonspecific binding [75] (4) Adapter ligation bias: such as adaptor contamination [41] (5) Reverse transcription bias [76] (6) PCR amplification bias [77] (7) Machine failure; for example, incorrect PCR cycling temperatures [17]
Sequencing and imaging (1) Experimenter bias: such as cluster crosstalk caused by overloading the flowcell [78] (2) Sequencing platform bias [65] (3) Sequence context: such as AT/GC enrichment [79] (4) Machine failure: such as failure of laser, hard drive, software, and fluidics