**2.2. DNA preparation**

Genomic DNA (gDNA) was isolated from peripheral blood leukocytes using the Wizard® Genomic DNA purification kit (Promega, Alexandria, NSW, Australia). DNA quality and concentration were assessed using the Nanodrop™ 1000 spectrophotometer (Thermo Scien‐ tific, Scoresby, Vic, Australia) that measures the purity of DNA by the ratio of absorbance of molecules at 260 and 280 nm. Samples with ratios between 1.8 and 2.0 were accepted for analysis whilst ratios lower than this may represent the presence of contaminants and these samples were not processed further [26]. At least, 250 ng of input gDNA was prepared per sample.

#### **2.3. Candidate gene identification and gene panel design**

An extensive literature search using public databases was performed to assemble an initial candidate gene list of all genes reasonably hypothesized to have an impact on platelet number and size (*n* = 173). A final list of candidate genes (*n* = 19) was derived by including those genes in which mutations were known to be definitively associated with IPNDs (predominantly, macrothrombocytopenia) and by excluding genes, which although known to result in throm‐ bocytopenia, could easily be identified by conventional and clinical methods characterized by distinct clinical phenotypes.

A TruSeq custom amplicon (TruSeq® Custom Amplicon Kit, Illumina Inc., Scoresby, Vic, Australia) specific for the target regions of the selected 19 genes (Table 1, *ACTN1, CD36, ETS1, F2R, FLI1, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, ITGA2, ITGA2B, ITGB1, ITGB3, MYH9, NBEAL2, P2RY12, RUNX1, TUBB1*) was designed as an entire custom pool using the webbased software tool, Illumina Design Studio (Illumina Inc.). This generated 201 gene targets that were either exons or gene regions that were split into 632 amplicons, each of approximately 250 base pairs (bps). There were no undesignable targets and a total coverage of 91% was predicted for the panel.

Following informed written consent, 20 ml of blood was taken from an antecubital vein and collected into EDTA tubes. This blood was easily transported, in some cases, over 1,000 km

A total of 95 patient DNA samples were analyzed. This included two internal controls for

32 male patients (mean age 37.4 years, range 18–92 years) and 44 female patients (mean age 38.7 years, range 18–79 years) were included in the NGS assay. The mean age of the cohort was 38.1 years (range 18–92 years). Sixteen de-identified DNA samples were received from

Phenotypic testing data were available for 59 (62.1%) individuals. This included platelet functional analysis (PFA) (*n* = 25, 26.0% of the cohort), light transmission aggregometry / whole blood impedance aggregometry (LTA/WBIA) (*n* = 39, 41.3% of the cohort), flow cytometry (*n* = 45, 47.8% of the cohort) and electron microscopy (*n* = 12, 13% of the cohort). These phenotypic test results suggested a diagnosis to a "pathway level", that is, a description to the level of the suspected defective biochemical pathway, in only 11 cases. Pathway orientated defects included, storage pool disorders (*n* = 3), platelet glycoprotein deficiency (*n* = 3), platelet signaling defects (*n* = 2), platelet secretion defects (*n* = 2) as well as α-granule disorder (*n* = 1).

Genomic DNA (gDNA) was isolated from peripheral blood leukocytes using the Wizard® Genomic DNA purification kit (Promega, Alexandria, NSW, Australia). DNA quality and concentration were assessed using the Nanodrop™ 1000 spectrophotometer (Thermo Scien‐ tific, Scoresby, Vic, Australia) that measures the purity of DNA by the ratio of absorbance of molecules at 260 and 280 nm. Samples with ratios between 1.8 and 2.0 were accepted for analysis whilst ratios lower than this may represent the presence of contaminants and these samples were not processed further [26]. At least, 250 ng of input gDNA was prepared per

An extensive literature search using public databases was performed to assemble an initial candidate gene list of all genes reasonably hypothesized to have an impact on platelet number and size (*n* = 173). A final list of candidate genes (*n* = 19) was derived by including those genes in which mutations were known to be definitively associated with IPNDs (predominantly, macrothrombocytopenia) and by excluding genes, which although known to result in throm‐ bocytopenia, could easily be identified by conventional and clinical methods characterized by

A TruSeq custom amplicon (TruSeq® Custom Amplicon Kit, Illumina Inc., Scoresby, Vic, Australia) specific for the target regions of the selected 19 genes (Table 1, *ACTN1, CD36, ETS1, F2R, FLI1, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, ITGA2, ITGA2B, ITGB1, ITGB3, MYH9, NBEAL2, P2RY12, RUNX1, TUBB1*) was designed as an entire custom pool using the webbased software tool, Illumina Design Studio (Illumina Inc.). This generated 201 gene targets that were either exons or gene regions that were split into 632 amplicons, each of approximately

which DNA-based diagnosis had previously been established by Sanger sequencing.

referring institutions for which no additional laboratory data were available.

**2.3. Candidate gene identification and gene panel design**

between diagnostic sites in Australia.

390 Next Generation Sequencing - Advances, Applications and Challenges

**2.2. DNA preparation**

distinct clinical phenotypes.

sample.


**Table 1.** Candidate gene list. OMIM, online Mendelian inheritance in man; AR, autosomal recessive; AD, autosomal dominant; XL, X-linked; nd, not defined, \*In progress (OMIM)
