Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.
We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.
Khattra
Jaswinder
J
Canada's Michael Smith Genome Sciences Centre, BC Cancer Research Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 4S6, Canada.
Delaney
Allen D
AD
Zhao
Yongjun
Y
Siddiqui
Asim
A
Asano
Jennifer
J
McDonald
Helen
H
Pandoh
Pawan
P
Dhalla
Noreen
N
Prabhu
Anna-Liisa
AL
Ma
Kevin
K
Lee
Stephanie
S
Ally
Adrian
A
Tam
Angela
A
Sa
Danne
D
Rogers
Sean
S
Charest
David
D
Stott
Jeff
J
Zuyderduyn
Scott
S
Varhol
Richard
R
Eaves
Connie
C
Jones
Steven
S
Holt
Robert
R
Hirst
Martin
M
Hoodless
Pamela A
PA
Marra
Marco A
MA
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation; D030561:Databases, Nucleic Acid; D053595:Embryonic Stem Cells / Q000737:chemistry; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D016678:Genome; D006801:Humans; D051379:Mice; D042282:Microdissection; D017422:Sequence Analysis, DNA; D012984:Software; D015027:Zebrafish / Q000235:genetics