Question

Oligo-dT(5’dTTTTTTTTTTTTTTTTTT3’) is used for the enrichment of polyA plus RNA, how does addition of UUUUU to the oligodT (dTTTTTTTTTTTTUUUUUU) increase the accuracy of identification of polyA site and plus RNA by Next Generation Sequencing

Is there still a possibility of getting a false positive? How so?

Answer 1

Other than priming at poly(A) tail of RNA molecules, oligo(dT) primers can also anneal to internal A-rich sequences, a phenomenon termed internal priming, leading to the generation of false pA peaks. Most of these fake pA peaks can be removed computationally based on the successive As in their downstream genomic sequences. However, such strategy would also lead to the systematical loss of real pA sites flanking A-rich sequences, which account for ∼ 8% of total pA sites in mouse transcriptome ]. Using PCR primers with additional Ts at the 3′ ends, the enriched cDNA molecules are reversely transcribed from poly(A)+ mRNA rather than from RNA molecules with internal A-rich sequences, thus reducing the effect of internal priming .

Disadvantages:-

The oligo(dT)-based methods have several shortcomings. Typically, when oligo(dT) primers are used in reverse transcription for 3′ end enrichment, the T-stretch of primers is retained during library construction, which has the benefit to preserve strand information, but can be problematic during sequencing. In Illumina sequencing platform, DNA molecules with homopolymers can probably lead to color imbalance and failure of cluster identification. Notably, base-calling quality significantly drops after sequencing through the T-stretch. This is likely due to the difference of sequencing starting point of molecules within the same cluster, a phenomenon called sequencing desynchronization .The drop of base-calling quality happens when sequencing starts from the termini corresponding to 3′ end of mRNA. Using fewer Ts in oligo(dT) primers can alleviate this defect; however, it will worsen the internal priming problem. Alternatively, libraries could also be sequenced from the termini corresponding to 5′ end of mRNA . In this way, the base-calling quality won’t be influenced by T-stretch. Nonetheless, only a proportion of reads can reach pA sites, leading to shallower sequencing depth at poly(A) termini, which sometimes would cause the failure in calling some real pA sites.