Comparative study of the different methods of small RNA high-throughput sequencing library preparation in human brain tissues / 中华老年医学杂志

ABSTRACT

Objective The aim of this study is to investigate the effects of the initial amount of total RNA input,adapter dilution and PCR amplification cycles on the results of small RNA library preparation and high-throughput sequencing.Methods Based on the sequencing reads number,the number of detected miRNAs and the accuracy of quantitative detection,we explored the effects of initial Total RNA,dilution ratio and PCR cycles on the quality of microRNA library preparation and high-throughput sequencing,respectively.Results For libraries preparation of the normal RNA input(1 μg),the adapter dilution combined with 22 PCR cycles could gain the best quality of sequencing.In the low-input libraries(10 ng),adapter dilution and increased PCR cycles would also improve the quality of sequencing,but as compared with the 1 μg library,there was lower correlation with microarray quantitative results in general.Conclusions The initial amount of RNAs input has the biggest effects on the quality of sequencing,and the quantity accuracy rate of low-input libraries is lower than the normal libraries generally.Increasing the PCR amplification cycles properly is indispensable to low-input libraries.