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 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 x 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).
Warrington
Janet A
JA
Affymetrix, Inc., Santa Clara, California, USA. Janet_Warrington@affymetrix.com
Shah
Nila A
NA
Chen
Xiyin
X
Janis
Michael
M
Liu
Chunmei
C
Kondapalli
Sangeetha
S
Reyes
Vivian
V
Savage
Michael P
MP
Zhang
Zhaomei
Z
Watts
Richard
R
DeGuzman
Maria
M
Berno
Anthony
A
Snyder
Jim
J
Baid
Jyoti
J
D001331:Automation; D001483:Base Sequence; D005260:Female; D005787:Gene Frequency; D014644:Genetic Variation / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D044469:Racial Groups / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D012984:Software
D012333:RNA, Messenger