Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

Table 2 Number of PCR cycles used for each sample

Sample	Clontech	Illumina	NEB	NEXTflex
100 ng Brain total RNA	13 cycles	NA	15 cycles	18 cycles
250 ng Brain total RNA	13 cycles	NA	15 cycles	18 cycles
500 ng Brain total RNA	11 cycles	NA	15 cycles	18 cycles
1000 ng Brain total RNA	10 cycles	11 cycles	15 cycles	18 cycles
1500 ng Brain total RNA	9 cycles	11 cycles	NA	18 cycles
2000 ng Brain total RNA	7 cycles	11 cycles	NA	18 cycles
300 ng of equimolar synthetic pool	7 cycles	11 cycles	15 cycles	18 cycles

Both batches of 1000 ng brain total RNA samples had the same number of cycles. We used the same number of cycles as suggested by each protocol for a range of input amounts. The brain samples were all derived from the same RNA extraction, purchased from Ambion of a 74-year-old Caucasian Female. The cause of death of this individual was respiratory failure. The Miltenyi Biotec miRXplore Universal Reference equimolar pool was used for the accuracy assessments

ISSN: 1471-2164