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

[hirschsprung disease]

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 .