Identification of an in vitro transcription-based artifact affecting oligonucleotide microarrays.
This study identified the widely used T7 in vitro transcription system as a major source of artifact in the tiling array data from nine eukaryotic genomes. The most affected probes contained a sequence motif complementary to the +1 to +9 initial transcribed sequence (ITS) of the T7-(dT)(24) primer. The abundance of 5' ITS cRNA fragments produced during target preparation was sufficient to drive undesirable hybridization. A new T7-(dT)(24) primer with a modified ITS was designed that shifts the artifactual motifs as predicted and reduces the effect of the artifact. A computational algorithm was generated to filter out the likely artifactual probes from existing whole-genome tiling array data and improve probe selection. Further studies of Arabidopsis thaliana were conducted using both T7-(dT)(24) primers. While the artifact affected transcript discovery with tiling arrays, it showed only a minor impact on measurements of gene expression using commercially available 'gene-only' expression arrays.
Nelson
David C
DC
Biotechnology Center and Biochemistry Department, University of Wisconsin, 425 Henry Mall, Madison, WI 53706, USA. msussman@wisc.edu
Wohlbach
Dana J
DJ
Rodesch
Matthew J
MJ
Stolc
Viktor
V
Sussman
Michael R
MR
Samanta
Manoj P
MP
D016477:Artifacts; D001483:Base Sequence; D009711:Nucleotides / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014158:Transcription, Genetic / Q000235:genetics*
D009711:Nucleotides