Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries

摘要: Background. Multiplexed sequencing is commonly performed on massively parallel short-read platforms such as Illumina, and the efficiency of library normalisation can affect quality output dataset. Although several approaches have been established, none are ideal for highly multiplexed due to issues cost and/or processing time. Methods. An inexpensive high-throughput quantification method has developed, based an adaptation melting curve assay. Sequencing libraries were subjected assay using Bio-Rad Laboratories CFX ConnectTM Real-Time PCR Detection System. The quantity was calculated through summation reduction relative fluorescence units between 86 95 °C. Results.PCR-enriched suitable this without pre-purification DNA. Short DNA molecules, which ideally should be eliminated from subsequent processing, differentiated target in a mixture basis differences temperature. Quantification results long sequences targeted correlated with those existing methods (R2 > 0.77), that observed MiSeq = 0.82). Discussion.The suggested performance described equivalent another recently reported bead-based method, BeNUS. However, costs considerably lower times shorter than other methods, suggesting greater suitability applications.