RNA fragmentation by RNase III: not completely random, leading to reduced complexity
Use 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 bias
RNA 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 ligases
Use adapters with random nucleotides at the extremities to be ligated [42]
PCR
(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]
