Indexed PCR primers induce template-specific bias in large-scale DNA sequencing studies

Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficiency. For studies that include a PCR step, this can be accomplished using primers that include an index sequence allowing sample origin to be determined after sequencing. The use of indexed primers assumes they behave no differently than standard primers; however, we found that indexed primers cause substantial template sequence-specific bias, resulting in radically different profiles of the same environmental sample. Likely the outcome of differential amplification efficiency due to primer-template mismatch, two indexed primer sets spuriously change the inferred sequence abundance from the same DNA extraction by up to 77.1%. We demonstrate that a double PCR approach alleviates these effects in applications where indexed primers are necessary.

大规模并行测序（Massively parallel sequencing）正迅速成为一种高效手段，可从遗传变异、基因表达乃至生态群落组成等多个层面量化生物多样性。然而在诸多应用场景中，单次测序运行产生的读段（reads）总量远超出单个样本的测序需求量，这促使研究者在单次运行中同时测序多个样本以提升测序效率。对于包含聚合酶链式反应（PCR, Polymerase Chain Reaction）步骤的研究，可借助带有索引序列（index sequence）的引物实现该目标，这类引物可在测序完成后用于判定样本来源。使用带索引引物（indexed primers）的前提是其性能与标准引物无显著差异；然而本研究发现，带索引引物会引发显著的模板序列特异性偏好性，导致同一环境样本的测序特征谱出现极大差异。该偏好性大概率源于引物与模板错配导致的扩增效率差异：两组带索引引物会使同一DNA提取物的推断序列丰度出现最高达77.1%的假性变化。本研究证实，在必须使用带索引引物的应用场景中，采用双PCR（double PCR）策略可有效缓解上述问题。