Gene expression analysis by signature pyrosequencing.
We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.
Agaton
Charlotta
C
Department of Biotechnology, KTH, Royal Institute of Technology SCFAB, Roslagstullsbacken 21, 106 91 Stockholm, Sweden.
Unneberg
Per
P
Sievertzon
Maria
M
Holmberg
Anders
A
Ehn
Maria
M
Larsson
Magnus
M
Odeberg
Jacob
J
Uhlén
Mathias
M
Lundeberg
Joakim
J
D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012107:Research Design; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein; D013755:Tetradecanoylphorbol Acetate