Because of the complexity of the MPS and MPSS techniques, the following summary is provided to describe the key aspects of the MPS and MPSS procedures:
- MPS is a technique often called next-generation sequencing (NGS) which provides high throughput DNA sequencing to determine the entire genomic sequence of a person or organism.
- The technique processes millions of DNA sequences in parallel rapidly sequencing whole genomes resulting in high resolution of specimens.
- MPS typically involves three basic steps:
- Step one is termed library preparation which prepares DNA or RNA for sequencing. This step yields DNA fragments which can be amplified and purified.
- Step two is the sequencing step which typically involves loading the DNA fragments onto a flow cell and then the fragments are amplified producing millions of copies of the single-stranded DNA. The amplified DNA templates are then sequenced simultaneousl in a sequencing instrument in a massively parallel fashion without the requirements for physical separation.
- Step three is the data analysis step which typically uses special software programs to identify nucleotides and analyze the sequencing data. The analysis allows for the identification of sequence alignment, variant calling, data visualization or interpretation.
- Since the MPS technology can be used to analyze millions of cfDNA fragments. it has been employed in several of the leading NIPT assays for the identification of various aneuploidies.
- MPSS is a related MPS technique which analyzes gene expression in a specimen by totaling the number of individual messenger-RNA (mRNA) produced in each gene.
- MPSS can analyze millions of cfDNA fragments and can sequence segments of cfDNA from both the mother and fetus. The technique analyzes complementary DNA (cDNA) which is synthesized from mRNA. The cDNAs are amplified to yield unique tags.which are attached to microbeads.where several rounds of sequence determinations are performed and a sequence pattern or signature is identified from each microbead. Each signature sequence is then analyzed, compared with all other signatures, and all identical signatures are counted. The number of chromosome counts compared to control values are perfomed and any excess of a chromosome (eg 21) would suggest a trisomy.
- MPSS does require analysis of very large numbers of DNA fragments per sample (about 25 million), which could potentially limit its clinical utility.
