Reference standards for next-generation sequencing

The analysis of next-generation sequencing (NGS) data is complex, owing to the breadth of sequences tested and the range of internal biases and errors. In a clinical context, this can lead to false positives and false negatives, and the potential for misdiagnosis.These errors and biases can be mitigated through the use of reference standards — materials with known characteristics that are crucial for test development, quality control and proficiency testing.Various reference standards have been developed for NGS, including well-characterized biological samples, synthetic controls and in silico data sets. Each approach has its own strengths and limitations.Despite recent progress in developing reference standards, several important challenges remain, including the need to establish the commutability of standards with patient samples.We consider an informed use of reference standards, along with associated statistical principles, to be essential for the rigorous analysis of NGS data. Furthermore, reference standards will have a key role in developing the next generation of sequencing technologies.