Fig. 4.

Mitigating consequences of oligo(dT) priming to internal A-rich stretches. (a) Genomically-encoded A/(G)-rich stretches internal to the transcripts can generate false positive pA sites, leading to incorrect calling of truncated ORFs or ncRNA processing sites. (b) Global read distribution across annotations for 37 °C versus 42 °C reverse transcription and addition of 42 °C pre-warmed reverse transcriptase. (c) Mis-priming of oligo(dT) to an A-rich “hot-spot” in the 25S rRNA at 37 °C (top) and 42 °C (bottom). Note the ribosomal RNA sequence is on the negative strand. (d) Nucleotide content 50 bp upstream and downstream of called pA sites at 37 °C (left) and 42 °C (right) for the indicated genomic regions. (e) Analysis of A/G richness downstream of pA sites for 37 °C (left) and 42 °C (right). The y-axis for the lefthand plots is a score calculated by the number of A’s in the 19 nucleotide downstream region (the region which would base-pair with the T-stretch of the oligo-d(T)V primer) with A in the first 6 bp downstream of the pA site given 2X weight. The score for the righthand plots contain additionally the number of G’s in the 19 nucleotide downstream region (where G is given half the score of A).