Consistent errors in first strand cDNA due to random hexamer mispriming.

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Random hexamer priming is a common method for complementary DNA (cDNA) synthesis in RNA sequencing (RNA-seq).
• Previous studies have noted sequence bias associated with random hexamers.
• Emerging evidence suggests mispriming events may contribute to sequence discrepancies between RNA and cDNA reads.

Purpose of the Study:

• To investigate random hexamer mispriming as a potential source of errors in RNA-seq data.
• To analyze sequence mismatch patterns in relation to random hexamer priming.

Main Methods:

• Analysis of two independent RNA-seq datasets derived from synthetic External RNA Controls Consortium (ERCC) spike-in sequences.
• Examination of first-strand cDNA synthesis using random hexamer priming on RNA templates.
• Characterization of mismatch errors in terms of position, nucleotide specificity, and thermodynamic stability.

Main Results:

• Consistent, position- and nucleotide-specific mismatch errors were observed in the first seven nucleotides of cDNA synthesized from RNA.
• Mismatch error distributions were similar across both analyzed datasets.
• Thermodynamically stable mismatches were found to be more prevalent, indicating a non-random error source.

Conclusions:

• RNA-DNA mispriming by specific random hexamers is strongly indicated as the cause of observed sequence errors in RNA-seq.
• The consistency and specificity of these mispriming errors necessitate careful consideration in downstream RNA-seq data analysis.
• Failure to account for mispriming errors may have significant implications for the accuracy of various biological applications.