title: Next-generation sequencing using a pre-designed gene panel for the molecular diagnosis of congenital disorders in pediatric patients.
abstract: BACKGROUND
  Next-generation sequencing (NGS) has revolutionized genetic research and offers enormous potential for clinical application. Sequencing the exome has the advantage of casting the net wide for all known coding regions while targeted gene panel sequencing provides enhanced sequencing depths and can be designed to avoid incidental findings in adult-onset conditions. A HaloPlex panel consisting of 180 genes within commonly altered chromosomal regions is available for use on both the Ion Personal Genome Machine (PGM) and MiSeq platforms to screen for causative mutations in these genes.
  
  
  METHODS
  We used this Haloplex ICCG panel for targeted sequencing of 15 patients with clinical presentations indicative of an abnormality in one of the 180 genes. Sequencing runs were done using the Ion 318 Chips on the Ion Torrent PGM. Variants were filtered for known polymorphisms and analysis was done to identify possible disease-causing variants before validation by Sanger sequencing. When possible, segregation of variants with phenotype in family members was performed to ascertain the pathogenicity of the variant.
  
  
  RESULTS
  More than 97% of the target bases were covered at >20×. There was an average of 9.6 novel variants per patient. Pathogenic mutations were identified in five genes for six patients, with two novel variants. There were another five likely pathogenic variants, some of which were unreported novel variants.
  
  
  CONCLUSIONS
  In a cohort of 15 patients, we were able to identify a likely genetic etiology in six patients (40%). Another five patients had candidate variants for which further evaluation and segregation analysis are ongoing. Our results indicate that the HaloPlex ICCG panel is useful as a rapid, high-throughput and cost-effective screening tool for 170 of the 180 genes. There is low coverage for some regions in several genes which might have to be supplemented by Sanger sequencing. However, comparing the cost, ease of analysis, and shorter turnaround time, it is a good alternative to exome sequencing for patients whose features are suggestive of a genetic etiology involving one of the genes in the panel.
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore, 229899, Singapore. Eileen.Lim.CP@kkh.com.sg.
authors.forename: Eileen C P
authors.initials: EC
authors.lastname: Lim
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore, 229899, Singapore. Maggie.Brett@kkh.com.sg.
authors.forename: Maggie
authors.initials: M
authors.lastname: Brett
authors.affiliation: Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore, 229899, Singapore. Angeline.Lai.HM@kkh.com.sg.|Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Graduate Medical School, Singapore, 169857, Singapore. Angeline.Lai.HM@kkh.com.sg.
authors.forename: Angeline H M
authors.initials: AH
authors.lastname: Lai
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore, 229899, Singapore. Lee.Siew.Peng@kkh.com.sg.
authors.forename: Siew-Peng
authors.initials: SP
authors.lastname: Lee
authors.affiliation: Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore, 229899, Singapore. tan.ee.shien@kkh.com.sg.|Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Graduate Medical School, Singapore, 169857, Singapore. tan.ee.shien@kkh.com.sg.
authors.forename: Ee-Shien
authors.initials: ES
authors.lastname: Tan
authors.affiliation: Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore, 229899, Singapore. saumya.s.jamuar@kkh.com.sg.|Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Graduate Medical School, Singapore, 169857, Singapore. saumya.s.jamuar@kkh.com.sg.
authors.forename: Saumya S
authors.initials: SS
authors.lastname: Jamuar
authors.affiliation: Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore, 229899, Singapore. ivy.ng@singhealth.com.sg.|Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Graduate Medical School, Singapore, 169857, Singapore. ivy.ng@singhealth.com.sg.
authors.forename: Ivy S L
authors.initials: IS
authors.lastname: Ng
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore, 229899, Singapore. tanec@bigfoot.com.|Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Graduate Medical School, Singapore, 169857, Singapore. tanec@bigfoot.com.
authors.forename: Ene-Choo
authors.initials: EC
authors.lastname: Tan
mesh_terms: D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D030541:Databases, Genetic; D059472:Exome; D005260:Female; D015723:Gene Library; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics; D020022:Genetic Predisposition to Disease; D015894:Genome, Human; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA