ABSTRACT

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.

摘要

高通量测序已成为研究 microRNA (miRNA) 表达的首选方法，但文库制备过程中引入的偏差已有报道。我们最近比较了六种市售试剂盒对合成 miRNA 和人类 RNA 的性能（灵敏度、可靠性、滴定响应和差异表达），其中文库制备由供应商进行。我们特此用另外两种常用试剂盒（NEBNext、NEXTflex）的数据补充本研究，这两种试剂盒的制造商最初拒绝参与。NEXTflex 表现出最高的灵敏度，这可能反映了其使用部分随机的接头序列，但总体性能低于 QIAseq 和 TailorMix 试剂盒。NEBNext 表现中等。我们重申，偏差是试剂盒特定的，这使得使用不同试剂盒生成的 miRNA 数据集的比较变得复杂。