Alternative expression analysis by RNA sequencing.
In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.
Griffith
Malachi
M
Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.
Griffith
Obi L
OL
Mwenifumbo
Jill
J
Goya
Rodrigo
R
Morrissy
A Sorana
AS
Morin
Ryan D
RD
Corbett
Richard
R
Tang
Michelle J
MJ
Hou
Ying-Chen
YC
Pugh
Trevor J
TJ
Robertson
Gordon
G
Chittaranjan
Suganthi
S
Ally
Adrian
A
Asano
Jennifer K
JK
Chan
Susanna Y
SY
Li
Haiyan I
HI
McDonald
Helen
H
Teague
Kevin
K
Zhao
Yongjun
Y
Zeng
Thomas
T
Delaney
Allen
A
Hirst
Martin
M
Morin
Gregg B
GB
Jones
Steven J M
SJ
Tai
Isabella T
IT
Marra
Marco A
MA
D017398:Alternative Splicing; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D030541:Databases, Genetic; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020224:Expressed Sequence Tags; D005472:Fluorouracil / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA / Q000379:methods*
D000964:Antimetabolites, Antineoplastic; D020033:Protein Isoforms; D012333:RNA, Messenger; D005472:Fluorouracil