Massively Parallel Sequencing (MPS) and Massively Parallel Signature Sequencing (MPSS)

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Massively Parallel Sequencing (MPS) and Massively Parallel Signature Sequencing (MPSS)

Massively parallel sequencing (MPS)
MPS is a technique that offers several high-throughput approaches to DNA sequencing. It is also often called next-generation sequencing (NGS). MPS is a high-throughput DNA sequencing method used to determine the entire genomic sequence of a person or organism. The method processes millions of DNA sequences in parallel rather than processing single amplicons. MPS typically uses miniaturized and parallelized platforms for the sequencing of millions of short reads (50-400 bases each) per instrument run resulting in a high resolution for each sample. The technology enables a broad range of testing applications including rapidly sequencing whole genomes, deeply sequencing of target regions, and using RNA sequencing for gene expression analysis, and sequencing cancer specimens to identify tumor subtypes.
MPS typically involves three basic steps:
  • Library Preparations: This step prepares DNA or RNA samples to enable them to be sequenced. Sequencing libraries are typically prepared by fragmenting a genomic DNA or complementary DNA (cDNA) sample and ligating specialized adapters to both fragment ends. The adapters contain complementary sequences that allow the DNA fragment to bind and then the fragments can be amplified and purified.
  • Sequencing: The sequencing step typically involves loading the libraries onto a flow cell and then placed on the sequencer. The clusters of DNA fragments are amplified resulting in millions of copies of single-stranded DNA. On most sequencing instruments, clustering occurs automatically. The amplified DNA templates are then sequenced simultaneously in a massively parallel fashion without the requirements for physical separation.
  • Data Analysis: After sequencing, many MPS instruments using special software programs identify nucleotides and analyze the sequencing data allowing for the identification of sequence alignment, variant calling, data visualization or interpretation.
The MPS technology can be used to analyze millions of cfDNA fragments. Therefore, it has been employed in several of the leading NIPT assays for the identification of various aneuploidies.
Massively parallel signature sequencing (MPSS)
Another MPS-related technique is massively parallel signature sequencing (MPSS) which analyzes the level of gene expression in a specimen by totaling the number of individual messenger-RNA (mRNA) molecules that are produced by each gene. MPSS is a random, sequencing technique that is capable of analyzing millions of cfDNA fragments. Therefore, the method can be used to sequence short segments of cfDNA from the mother and the fetus and then assigns them to specific chromosomes. After comparing the number of chromosome- counts to a control value of other chromosomes, any excess of a particular chromosome (eg, 21) would suggest a trisomy.
In the MPSS procedure, tagged polymerase chain reaction (PCR) products produced from complementary DNA (cDNA), which is the DNA synthesized from a mRNA, are amplified so that each corresponding mRNA molecule will yield about 100,000 PCR products with unique tags. The tags are used to attach the PCR products to microbeads. Several rounds of sequence determinations are performed, and a sequence pattern or signature is identified from each microbead. The process is performed in parallel with about 1 million sequence signatures produced per overall assay. Each signature sequence is then analyzed, compared with all other signatures, and all identical signatures are counted. The level of expression of any single gene is then calculated. MPSS does require analysis of very large numbers of DNA fragments per sample (about 25 million), which could potentially limit its clinical utility.