title: Targeted next-generation sequencing on Hirschsprung disease: a pilot study exploits DNA pooling.
abstract: To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects.
authors.affiliation: Department of Surgery, The University of Hong Kong, Hong Kong, SAR, China; Department of Psychiatry, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Gui
authors.forename: Jessie Yunjuan
authors.initials: JY
authors.lastname: Bao
authors.forename: Clara Sze-Man
authors.initials: CS
authors.lastname: Tang
authors.forename: Man-Ting
authors.initials: MT
authors.lastname: So
authors.forename: Diem-Ngoc
authors.initials: DN
authors.lastname: Ngo
authors.forename: Anh-Quynh
authors.initials: AQ
authors.lastname: Tran
authors.forename: Duc-Hau
authors.initials: DH
authors.lastname: Bui
authors.forename: Duy-Hien
authors.initials: DH
authors.lastname: Pham
authors.forename: Thanh-Liem
authors.initials: TL
authors.lastname: Nguyen
authors.forename: Amy
authors.initials: A
authors.lastname: Tong
authors.forename: Si
authors.initials: S
authors.lastname: Lok
authors.forename: Pak-Chung
authors.initials: PC
authors.lastname: Sham
authors.forename: Paul Kwong-Hang
authors.initials: PK
authors.lastname: Tam
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Cherny
authors.forename: Maria-Mercè
authors.initials: MM
authors.lastname: Garcia-Barcelo
mesh_terms: D044466:Asian People / Q000235:genetics*; D017615:Enteric Nervous System / Q000502:physiology; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006627:Hirschsprung Disease / Q000235:genetics*; D006801:Humans; D008297:Male; D010865:Pilot Projects; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology
keywords: CDS; HSCR; RainDance; rare variant; targeted resequencing