**5. Appendix**

sequencing was also performed for *GP1BA* and *GP1BB* in cases in which phenotypic details had been provided by the referring clinician and where confident exclusion of a variant in those genes was necessary. Sanger sequencing performed over these regions did not detect additional mutations. Only a single false positive result was confirmed by Sanger sequencing (*RUNX1*, stop/gain, c.966T>G). This suggested good platform specificity. The question as to whether confirmatory Sanger sequencing need be performed is debated in the literature [39, 67]. Proponents argue that it is required to confirm a diagnosis as well as remove incorrect calls introduced by experimental errors. Whereas, opponents argue, in the setting where the NGS platform performance metrics have been established to be comparable to Sanger sequencing performance measures, a strategy dictated by the degree of coverage per nucleo‐ tide be adopted. Suggesting that parallel Sanger sequencing need not be performed as long as the coverage is >30 times per nucleotide at that genomic target, adding that confirmatory testing be performed where coverage is less than 20 times, and be determined by visual inspection with coverage between 20 and 30 times. Authors commented that the laboratory may also simply elect to exclude the target from the report if Sanger sequencing is not

An important aspect of the post-analytical process is the timely provision of a genomic test report. In the setting of inherited platelet disorders, a false negative interpretation may lead to a falsely conservative bleeding prophylactic strategy at the time of surgery, in turn, placing the individual at a potentially increased risk of bleeding. A false positive result, on the other hand, may cause undue stress to the individual and their family. A genomic test report was therefore carefully and consistently structured taking into consideration recommendations from professional bodies such as the RCPA [65] and ACMG [68]. The report (Appendix 1) contained a summary of the genes analyzed and reflected the scope and limitation of the assay and indicated the context in which the test was performed. A clear, succinct, interpretative comment was made regarding the detected variant. This indicated whether or not the detected variant was associated with the clinical phenotype and highlighted variants of uncertain significance. The body of the report detailed, in a structured format (see materials and methods), any detected pathogenic or clinically relevant variants and whether these had been previously described. An interpretation on the significance of the detected variant was supported by relevant references where possible, and recommendations regarding additional validation tests and /or genetic counseling and clinical screening were provided. Following the main body of the report, DNA variants that were considered to be non-pathogenic were listed. The report was concluded by a description of the test method and limitations thereof.

In conclusion, our study has demonstrated the potential to successfully diagnose inherited macrothrombocytopenia in cases that remained uncharacterized by traditional phenotypic approaches. Optimization of this format will provide patients an opportunity for a "one stop, one step" testing platform that is cost-effective and not affected by the pre-analytical variables that hinder current testing methods based on functional analysis of platelets. However, the translation of NGS from a powerful research tool into the clinical laboratory will require cooperation from international groups to establish best practice, quality and reporting standards

performed despite low coverage [39].

400 Next Generation Sequencing - Advances, Applications and Challenges

**Test performed**: Candidate gene array of 19 genes (*ACTN1, CD36, F2R, FLI1, ETS1, GATA1, GFI1b, GP1BA, GP1BB, GP6, GP9, ITGA2, ITGA2B, ITGB1, ITGB3, MYH9, NBEAL2, P2RY12, RUNX1, TUBB1)* using the Illumina MiSeq next-generation sequencing platform.
