Pan-genome isolation of low abundance transcripts using SAGE tag.
The SAGE (serial analysis of gene expression) method is sensitive at detecting the lower abundance transcripts. More than a third of human SAGE tags identified are novel representing the low abundance unknown transcripts. Using the GLGI method (generation of longer 3' EST from SAGE tag for gene identification), we converted 1009 low-copy, human X chromosome-specific SAGE tags into 10210 3' ESTs. We identified 3418 unique 3' ESTs, 46% of which are novel and originated from the lower abundance transcripts. However, nearly all 3' ESTs were mapped to various regions across the genome but not X chromosome. Detailed analysis indicates that those 3' ESTs were isolated by SAGE tag mis-priming to the non-parent transcripts. Replacing SAGE tags with non-transcribed genomic DNA tags resulted in poor amplification, indicating that the sequence similarity between different transcripts contributed to the amplification. Our study shows the prevalence of novel low abundance transcripts that can be isolated efficiently through SAGE tags mis-priming.
Kim
Yeong Cheol
YC
Center for Functional Genomics, Division of Medical Genetics, Department of Medicine, ENH Research Institute, Northwestern University, Evanston, IL 60201, USA.
Jung
Yong-Chul
YC
Xuan
Zhenyu
Z
Dong
Hui
H
Zhang
Michael Q
MQ
Wang
San Ming
SM
D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D041321:Chromosomes, Human, X / Q000235:genetics; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000235:genetics*
D012333:RNA, Messenger