New developments in high-throughput resequencing and variation detection using high density microarrays

✍ Scribed by Janet A. Warrington; Nila A. Shah; Xiyin Chen; Michael Janis; Chunmei Liu; Sangeetha Kondapalli; Vivian Reyes; Michael P. Savage; Zhaomei Zhang; Richard Watts; Maria DeGuzman; Anthony Berno; Jim Snyder; Jyoti Baid

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 517 KB
• Volume: 19
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/humu.10075

✦ Synopsis

For the Mutation Detection 2001 Special Issue

We developed a high-throughput method for resequencing for single nucleotide polymorphism (SNP) discovery using high-density microarrays. Over the two-year course of this study a number of improvements in sample preparation methods, hybridization assay, array handling, and analysis method were developed and implemented. DNA from 40 unrelated individuals of three different ethnic origins was amplified, labeled, and hybridized to arrays designed with probes representing genomic, coding, and regulatory regions. Protocol improvements including the use of long PCR and semi-automation reduced labeling and fragmentation costs by 33%. Automation improvements include the development of a scanner autoloader for arrays, a faster array wash station, and a linked laboratory tracking and data management system. Validation of a smaller feature size, 20 ´ 24 microns, allowed the simultaneous screening of 30-kb sense and 30kb antisense DNA on each microarray, increasing throughput to 1.4 Mb per day per two laboratory personnel. More than 15,000 SNPs were identified in 8.3 Mb of the human genome using high-density resequencing and variation detection arrays (microarrays).