Use RNAz to further verify whether any of those false positives filtered by order-1 method and those not filtered by order-1 method is ncRNA.

We use another popular software RNAz [8] to further evaluate the candidates filtered by our order-1 method. Only 3% are estimated to be ncRNAs by RNAz. We perform a similar test on the candidates that kept by our order-1 method. About 18–33% are estimated to be ncRNAs by RNAz. Although RNAz may not give an accurate result, it provides some evidence that most of the filtered candidates may be false positives. On the other hand, the candidates that kept by our order-1 method but cannot be confirmed by RNAz should be further evaluated.