Consistent errors in first strand cDNA due to random hexamer mispriming

TY - JOUR

T1 - Consistent errors in first strand cDNA due to random hexamer mispriming

AU - Van Gurp, T.P.

AU - McIntyre, L.M.

AU - Verhoeven, K.J.F.

N1 - Reporting year: 2013 Metis note: 5497; TE Data archiving: no data, sequencing data

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - international

U2 - 10.1371/journal.pone.0085583

DO - 10.1371/journal.pone.0085583

M3 - Article

SN - 1932-6203

VL - 8

JO - PLoS One

JF - PLoS One

IS - 12

M1 - e85583

ER -