Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias.

本研究展示了一种基于新型高效且可灵活适配的条形码策略——扩增后连接介导（Post Amplification Ligation-Mediated，PALM）——实现RNA与DNA文库无偏倚多重深度测序的方法。PALM条形码标记在文库制备的最终阶段完成，可消除潜在的条形码诱导偏倚，且能够灵活合成任意数量所需的条形码。本研究对经PALM标记的微小RNA（micro RNA，miRNA）与DNA参考样本进行测序，并与采用Illumina TruSeq条形码策略制备的同一批参考样本对比，评估了条形码诱导的定量偏倚。Illumina TruSeq小RNA测序策略在聚合酶链式反应（PCR）阶段使用带差异化条形码的引物引入条形码，而TruSeq DNA测序策略则在PCR前通过连接带差异化条形码的接头引入条形码。结果显示，无论采用PALM还是TruSeq样本制备方法，带差异化条形码的miRNA与DNA样本之间几乎不存在偏倚。本研究还采用PCR前条形码连接的方式对miRNA参考样本进行多重标记，该条形码策略会导致显著的偏倚。