RNA-seq 5-prime to 3-prime Bias

Source of 3-prime Bias in PolyA-enriched RNA-seq

Cited from Figure 8: The use of 3′ bias as a quality control assay for cDNA.

"Total (bulk) RNA derived from tissue is confirmed to have a high RIN score before isolation of nuclei. Partial degradation of the RNA might occur during the preparation of nuclei by Dounce homogenization (nuclei prep) or FACS of the individual nuclei. If the mRNA is degraded by hydrolysis, shearing or RNases, truncated mRNA species could be created, and those containing the polyA sequence at the 3′ end of the transcripts might produce cDNA. This would generate greater RNA-seq coverage of the 3′ end of transcripts (3′-bias) compared with the high-quality bulk RNA."

======================================

Cited from NGS Quality Control in RNA Sequencing- Some Free Tools

To visualise 5-prime or 3-prime bias, use the tools of Picard or RSeQC.

======================================

Cited from How to understand median 5 prime to 3 prime bias ratio from Picard?

It is up to the analysis software to deal with 5-prime or 3-prime bias.

======================================

Cited from  Salmon Doc

"--seqBias" to learn sequence bias

Salmon uses a variable-length Markov Model (VLMM) to model the sequence specific biases at both the 5’ and 3’ end of sequenced fragments. This methodology generally follows that of Roberts et al. [2], though some details of the VLMM differ.