Review Article

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

Table 3

Sources of bias in RNA-seq library preparation and suggestions for improvement.

DescriptionSuggestion for improvement

mRNA enrichment
3-end capture bias that is introduced during poly (A) enrichment in RNA sequencingUse rRNA depletion [81].
RNA fragmentation by RNase III: not completely random, leading to reduced complexityUse chemical treatment (e.g., zinc) rather than RNase III for RNA fragmentation [31]
Intact RNAs can be reverse transcribed to cDNA by reverse transcriptase, then which was fragmented by mechanical or enzymatic methods [82]
Priming bias
Random hexamer priming biasRNA is not converted to dscDNA using random priming, instead of sequencing adapters that are ligated directly onto RNA fragments [39]
A read count reweighing scheme was proposed that adjusts for the bias and makes the distribution of reads more uniform [40]
Adapter ligation
Adapter ligation bias: due to substrate preferences of T4 RNA ligasesUse adapters with random nucleotides at the extremities to be ligated [42]
(1) Bias due to preferential amplification of with neutral GC%
(2) The number of cycles of high PCR amplification
Use Kapa HiFi rather than Phusion polymerase [49]
For extremely AT/GC-rich genomes, use the PCR additive TMAC or betaine, or lower extension temperatures and extended denaturation times [17, 48]
Reduce the number of cycles of amplification [55]
For the amplification of minute quantities of genomic DNA (single cell), use MDA rather than PCR [83]
A large number of starting material, use amplification-free PCR [47]