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

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 devel...

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Bibliographic Details
Published in:Human mutation 2002-04, Vol.19 (4), p.402-409
Main Authors: Warrington, Janet A., Shah, Nila A., Chen, Xiyin, Janis, Michael, Liu, Chunmei, Kondapalli, Sangeetha, Reyes, Vivian, Savage, Michael P., Zhang, Zhaomei, Watts, Richard, DeGuzman, Maria, Berno, Anthony, Snyder, Jim, Baid, Jyoti
Format: Article
Language:English
Subjects:
Automation
Base Sequence
Continental Population Groups - genetics
Female
Gene Frequency
Genetic Variation - genetics
Genome, Human
genotype
Humans
Male
microarray
mutation detection
Oligonucleotide Array Sequence Analysis - instrumentation
Oligonucleotide Array Sequence Analysis - methods
Polymorphism, Single Nucleotide - genetics
RNA, Messenger - analysis
RNA, Messenger - genetics
Sequence Analysis, DNA - instrumentation
Sequence Analysis, DNA - methods
SNPs
Software
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Summary: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 30‐kb 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). Hum Mutat 19:402–409, 2002. © 2002 Wiley‐Liss, Inc.
ISSN:1059-7794
1098-1004
DOI:10.1002/humu.10075