- Massively parallel signature sequencing
-
Massive parallel signature sequencing (MPSS) is a sequenced based approach that can be used to identify and quantify mRNA transcripts present in a sample similar to serial analysis of gene expression (SAGE) but the biochemical manipulation and sequencing approach differ substantially.
MPSS allows mRNA transcripts to be identified through the generation of a 17-20 bp (base pair) signature sequence adjacent to the 3’-end of the 3’- most site of the designated restriction enzyme (commonly Sau3A or DpnII). Each signature sequence is cloned onto one of a million microbeads. The technique ensures that only one type of DNA sequence is on a microbead. So if there are 50 copies of a specific transcript in the biological sample, these transcripts will be captured onto 50 different microbeads, each bead holding roughly 100 000 amplified copies of the specific signature. The microbeads are then arrayed in a flow cell for sequencing and quantification. The sequence signatures are deciphered by the parallel identification of four bases by hybridization to fluorescently labeled encoders (Figure 5). Each of the encoders has a unique label which is detected after hybridization by taking an image of the microbead array. The next step is to cleave and remove that set of four bases and reveal the next four bases for a new round of hybridization to encoders and image acquisition. The raw output is a list of 17-20 bp signature sequences, that can be annotated to the human genome for gene identification. The longer tag sequence confers a higher specificity than the classical SAGE tag of 9-10 bp. The level of unique gene expression is represented by the count of transcripts present per million molecules, similar to SAGE output. A significant advantage is the larger library size compared with SAGE. An MPSS library typically holds 1 million signature tags, which is roughly 20 times the size of a SAGE library. Some of the disadvantages related to SAGE apply to MPSS as well, such as loss of certain transcripts due to lack of restriction enzyme recognition site and ambiguity in tag annotation. The high sensitivity and absolute gene expression certainly favors MPSS. However, the technology is only available through Lynxgen Therapeutics, Inc. (then Solexa Inc till 2006 and then Illumina). Massively Parallel Signature Sequencing (MPSS) Introduction Massively Parallel Signature Sequencing (MPSS) is an open-ended platform that analyses the level of gene expression in a sample by counting the number of individual mRNA molecules produced by each gene.
Tagged PCR products produced from cDNA are amplified so that each corresponding mRNA molecule gives ~100,000 of PCR products with a unique tag. Tags are used to attach the PCR products to microbeads. After several rounds of ligation-based sequence determination using the type IIs restriction endonuclease BbvI, a sequence signature of ~16-20 bp is identified from each bead; routinely, 17 bp of high quality sequence is obtained. This is performed in parallel, and approximately 1,000,000 sequence signatures are obtained per experiment.
Each signature sequence (MPSS tag) in a MPSS dataset is analyzed, compared with all other signatures and all identical signatures are counted. The level of expression of any single gene is calculated by dividing the number of signatures from that gene by the total number of signatures for all mRNAs present in the dataset. MPSS datasets are additive in nature, which means that datasets from multiple analyses with the same starting mRNA sample can be combined.
MPSS has routine sensitivity at a level of a few molecules of mRNA per cell, and the datasets are in a digital format that simplifies the management and analysis of the data.
How it works
cDNA fragments are cloned onto micro-beads using the Lynx Megaclone technology. Starting with ome million mRNA molecules from a particular cell or tissue sample, Megaclone will produce one million beads, each containing 100,000 cloned copies of cDNA from each mRNA molecule. All molecules are covalently attached to the micro-beads at their poly(A) ends, so the DpnII end is available for the sequencing reactions.
Probe Sample QueriesSearch Text Probes MPSS tag[probe type]
References » Reinartz J et al. Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms. Brief Funct Genomic Proteomic. 2002 Feb;1(1):95-104. PMID: 15251069
» Brenner S et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol. 2000 Jun;18(6):630-4. Erratum in: Nat Biotechnol 2000 Oct;18(10):1021. PMID: 10835600
» Torres TT, Metta M, Ottenwälder B, Schlötterer C. Gene expression profiling by massively parallel sequencing. Genome Res. 2007 Nov 21 PMID: 18032722
References
Further reading
- Brenner, Sydney; Johnson, Maria; Bridgham, John; Golda, George; Lloyd, David H.; Johnson, Davida; Luo, Shujun; McCurdy, Sarah et al. (2000). "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays". Nature Biotechnology 18 (6): 630–4. doi:10.1038/76469. PMID 10835600.
- Nygaard, V; Hovig, E (2009). "Methods for quantitation of gene expression". Frontiers in bioscience : a journal and virtual library 14: 552–69. PMID 19273085.
External links
Categories:
Wikimedia Foundation. 2010.
