Targeted next-generation sequencing on hirschsprung disease: A pilot study exploits DNA pooling

Hongsheng Gui, Jessie Yunjuan Bao, Clara Sze Man Tang, Man Ting So, Diem Ngoc Ngo, Anh Quynh Tran, Duc Hau Bui, Duy Hien Pham, Thanh Liem Nguyen, Amy Tong, Si Lok, Pak Chung Sham, Paul Kwong Hang Tam, Stacey S. Cherny, Maria Mercè Garcia-Barcelo

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)381-387
Number of pages7
JournalAnnals of Human Genetics
Issue number5
StatePublished - Sep 2014
Externally publishedYes


  • CDS
  • HSCR
  • RainDance
  • Rare variant
  • Targeted resequencing


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