Differences between Methods
Given that both methods replicate at similar levels and Cufflinks finds more asQTLs, one can make the argument that this could be the method of choice. However, almost half of the asQTLs that are discovered with Altrans are unique to Altrans. Although the methodology in identifying splicing QTLs in the original Geuvadis analysis differs significantly from the process described here, we also checked the asQTL gene level overlap between the published lists of splicing QTLs7 and the ones identified here (Figure S3). We find that Altrans detects 258 out of the 620 asQTLs identified in the Europeans in the original study, and Cufflinks finds 348 overlapping asQTLs. The union of both methods used here identifies 395 genes as significant asQTLs out of the 620 in the original discovery. In the African population, the overlap proportions are similar, with Altrans finding 16 out of 83 asQTLs as also significant, whereas Cufflinks finds 35 common genes, and the union of Altrans and Cufflinks overlaps with 38 asQTLs in the original study. This is a confirmation of the complementary nature of asQTL discovery methods.
We investigated the Altrans-specific asQTLs further. First we find that the majority of the Altrans-specific asQTLs originate from links between exons that are not annotated in the GENCODE v.12 annotation and therefore were never tested by Cufflinks (89% and 83% not annotated for Europeans and Africans, respectively; Figure S3). Next, we assessed whether Altrans-specific discoveries replicate, and to do so we tested the Altrans-specific discoveries originating from the 91 CEU individuals in the remaining Europeans, and these associations achieve a π1 statistic of 93%, indicating a high true positive rate in Altrans-specific asQTLs (Figure S4A). We also estimate that 63% of the Altrans-specific asQTLs in Europeans are replicated in Africans and 95% of the African Altrans-specific asQTLs are replicated in Europeans.
Moreover, we compared the types of splicing events that are found to be significant by both methods (Figure S5) and observed that there are differences between the two methods. The majority (66%) of the signal that Altrans captures is due to exon skipping events followed by alternative 5′ and 3′ UTRs (15% and 11%, respectively). In comparison, Cufflinks has a more uniform distribution of significant event types, with the most common being alternative 5′ UTR (23%), followed by exon skipping (15%) and alternative first exons (14%). This difference in types of significant splicing events each method finds highlights their relative merits in identifying different types of splicing events and is one of the reasons for method-specific significant results. We have tested whether the exon skipping events identified by Altrans replicate between CEU discovery and remaining Europeans, and across populations, and we achieve high π1 values of 98% for CEU discovery replicated in remaining Europeans (Figure S4B), 70% for Europeans replicated in Africans, and 96% in Africans replicated in Europeans, which confirms that these events are enriched for true positives.