Bias in RNA-seq Library Preparation: Current Challenges and Solutions

Table 1

Sources of main bias in RNA-seq.

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Bias sources

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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

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