Massive parallel signature sequencing (MPSS) is a procedure that is used to identify and quantify mRNA transcripts, resulting in data similar to serial analysis of gene expression (SAGE), although it employs a series of biochemical and sequencing steps that are substantially different.
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High throughput sequencing
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DNA sequencing methods
Transcription
DNA sequencing is the process of making a choice on the precise order of nucleotides inside a DNA molecule. It involves any system or science that's used to investigate the order of the four bases adenine, guanine, cytosine, and thymine in a strand of DNA. The arrival of speedy DNA sequencing ways has extensively accelerated biological and medical research and discovery. Talents of DNA sequences has end up quintessential for basic biological research, and in numerous utilized fields akin to clinical diagnosis, biotechnology, forensic biology, virology and organic systematics. The fast speed of sequencing attained with ultra-modern DNA sequencing science has been instrumental in the sequencing of complete DNA sequences, or genomes of numerous varieties and species of life, together with the human genome and different whole DNA sequences of many animal, plant, and microbial species. An example of the results of automatic chain-termination DNA sequencing. The first DNA sequences had been bought within the early Nineteen Seventies by tutorial researchers utilizing laborious ways headquartered on two-dimensional chromatography. Following the development of fluorescence-headquartered sequencing approaches with automated evaluation, DNA sequencing has turn out to be simpler and orders of magnitude turbo. Up to date advances in DNA sequencing have revolutionized the area of genomics, making it possible for even single study companies to generate massive amounts of sequence data very quickly and at a appreciably decrease fee. These excessive-throughput sequencing applied sciences make deep transcriptome sequencing and transcript quantification, whole genome sequencing and resequencing available to many more researchers and projects. Nevertheless, whilst the cost and time were broadly decreased, the error profiles and barriers of the new systems vary significantly from these of previous sequencing technologies. The decision of an appropriate sequencing platform for unique types of experiments is an primary consideration, and requires a specified understanding of the technologies to be had; together with sources of error, error rate, as good as the speed and price of sequencing. We overview the crucial concepts and compare the issues raised by means of the current high-throughput DNA sequencing applied sciences. We analyze how future tendencies may overcome these limitations and what challenges stay.
How it works
MPSS is a method for determining expression levels of mRNA by counting the number of individual mRNA molecules produced by each gene. It is "open ended" in the sense that the identity of the RNAs to be measured are not pre-determined as they are with gene expression microarrays.
A sample of mRNA are first converted to complementary DNA (cDNA) using reverse transcriptase, which makes subsequent manipulations easier. These cDNA are fused to a small oligonucleotide "tag" which allows the cDNA to be PCR amplified and then coupled to microbeads. After several rounds of sequence determination, using hybridization of fluorescent labeled probes, a sequence signature of ~16–20 bp is determined from each bead. Fluorescent imaging captures the signal from all of the beads, while affixed to a 2-dimensional surface, so DNA sequences are determined from all the beads in parallel. There is some amplification of the starting material so, in the end, approximately 1,000,000 sequence reads are obtained per experiment.[1]
Overview
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 sequence. 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.
Comparison with SAGE
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).
References
- ^ Schuler Group. "Massively Parallel Signature Sequencing (MPSS)".
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. S2CID 13884154.
- Nygaard, V; Hovig, E (2009). "Methods for quantitation of gene expression". Frontiers in Bioscience. 14 (14): 552–69. doi:10.2741/3262. PMID 19273085.
- Reinartz, J; Bruyns, E; Lin, JZ; et al. (February 2002). "Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms". Brief Funct Genomic Proteomic. 1: 95–104. doi:10.1093/bfgp/1.1.95. PMID 15251069.
- Torres, TT; Metta, M; Ottenwälder, B; Schlötterer, C (January 2008). "Gene expression profiling by massively parallel sequencing". Genome Res. 18 (1): 172–7. doi:10.1101/gr.6984908. PMC 2134766. PMID 18032722.
External links
This page was last edited on 12 August 2023, at 13:25