Effects of duplicated mapped read PCR artifacts on RNA-seq differential expression analysis based on qRNA-seq

Best practices to handling duplicated mapped reads in RNA-seq analyses has long been discussed but a gold standard method has yet to be established, as such duplicates could originate from valid biological transcripts or they could be PCR-related artifacts. Here we used the NEXTflex™qRNA-SeqTM(aka Molecular Indexing™) technology to identify PCR duplicates via the random attachment of unique molecular labels to each cDNA molecule prior to PCR amplification. We found that up to 64.3% of the single end and 19.3% of the mouse paired end duplicates originated from valid biological transcripts rather than PCR artifacts. For single end reads, either removing or retaining all duplicates resulted in a substantial number of false positives (up to 47.0%) and false negatives (up to 12.1%) in the sets of significantly differentially expressed genes. For paired end reads, only the alignment retaining all duplicates resulted in a substantial number of false positives. This is the first effort to evaluate the performance of qRNA-seq using ‘real-world’ biomedical samples, and we found that PCR duplicate identification provided minor benefits for paired end reads but greatly improved the sensitivity and specificity in the determination of the significantly differentially expressed genes for single end reads.

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of theBordetellaphage DGR is primed by an adenine residue inTRRNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but isVR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage ofTRRNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation.