**Author details**

**Recommendations**: The pathogenicity of detected candidate variants should be validated independently by Sanger sequencing. Where necessary, the functional significance of these variants should be confirmed independently by appropriate biological assays to replicate the

MYH9-related disorders have an autosomal dominant inheritance. Genetic counselling is recommended for this individual and their family. Family screening may be appropriate after

*NBEAL2*, Heterozygous, c.1531C>G (p.Arg511Gly), Exon 13, rs11720139, likely **non-patho‐ genic**. *GP6*, Homozygous, c.691G>A (p.Ala231Thr), Exon 6, rs2304167, likely **non-pathogen‐ ic**. *MYH9*, Heterozygous, c.4876A>G (p.IIe1626Val), Exon 34, rs2269529, likely **non-**

A TruSeq custom amplicon specific for the target regions of 19 genes, *ACTN1, CD36, F2R, FLI1, ETS1, GATA1, GFI1b, GP1BA, GP1BB, GP6, GP9, ITGA2, ITGA2B, ITGB1, ITGB3, MYH9, NBEAL2, P2RY12, RUNX1, TUBB1* was designed using Illumina design studio (Illumina, Inc, San Diego, CA, USA). Next-generation sequencing was performed using the MiSeq Illumina sequencer platform (Illumina, Inc.). Obtained sequences were aligned to the reference genome (GRCh37/hg19) using MiSeq reporter software (Illumina, Inc.) and the genomic datasets viewed using the Integrative Genomics viewer (IGV) (www.broadinstitute.org/igv/). Variant calls were generated using ANNOVAR software (http://www.openbioinformatics.org/ annovar) with an acceptance threshold Q-score of 30, corresponding to a 1:1000 error rate. Sanger sequencing was performed to provide data for bases with insufficient coverage. The University of California, Santa Cruz (UCSC), genome browser (http://genome.ucsc.edu) was used for variant analysis and variants were cross-checked against databases including the NHLBI-extended sequencing project (ESP), 1000 genomes project database and the Database of Single-Nucleotide Polymorphisms (dbSNP). Bioinformatic tools (SIFT, PolyPhen-2 and Mutation taster) were used to predict variant effects on protein structure and function in the

**Limitations:** Overall gene coverage was 97% using this format. Therefore, it is possible that the genomic region where a disease causing mutation exists in the proband was not captured

It is also possible that a particular genetic mutation was not recognised as the underlying cause of the genetic disorder due to incomplete scientific knowledge of the impact of all variants at

phenotype of this patient.

**pathogenic**.

**Test method**:

appropriate genetic counselling.

**DNA variants detected of unlikely clinical significance:**

402 Next Generation Sequencing - Advances, Applications and Challenges

cases of variants lacking published literature.

and therefore was not detected.

this point in the literature.

An example of a NGS report.

**Reported by:**

David J. Rabbolini1,2, Marie-Christine Morel Kopp1,2, Sara Gabrielli1,2, Qiang Chen1,2, William S. Stevenson1,2 and Christopher M. Ward1,2\*

\*Address all correspondence to: cward@med.usyd.edu.au

1 Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Sydney, Australia

2 Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, Australia
