ArticlePloS one2013
Consistent errors in first strand cDNA due to random hexamer mispriming.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed.
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- Combining bioinformatics and conventional PCR optimization strategy for one-time design of high-specificity primers for WRKY gene family using unigene database.Molecular biology reports · 2019Article
- Mapping the dsRNA World.Cold Spring Harbor perspectives in biology · 2019Review
- A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing.FASEB bioAdvances · 2019Article
- Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma.Journal of virology · 2019Article
- A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing.FASEB bioAdvances · 2019Article
- Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies.RNA (New York, N.Y.) · 2018Article
- Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken.BMC genomics · 2018Article
- Identification of RNA-binding protein targets with HyperTRIBE.Nature protocols · 2018Article
- Intergenerational environmental effects: functional signals in offspring transcriptomes and metabolomes after parental jasmonic acid treatment in apomictic dandelion.The New phytologist · 2018Article
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.
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