massively parallel sequencing (MPS)

choi

(1) Next-Generation Sequencing; An introduction to NGS techniques. Slipcademy, undated (in its category "Bioinformatics")
https://snipcademy.com/ngs-techniques

Quote:

"It wasn't until pyrosequencing and other NGS [Next-Generation Sequencing] techniques that allowed for a price drop to $1000 * * *

"Resequencing vs. de novo sequencing[:] * * * Resequencing is the term for sequencing an organism that has already been sequenced. We only need to align our reads to a reference genome. Thus, our reads need only be a few hundred base pairs long. Many Next-Generation Sequencing platforms provide short reads that can be aligned to a reference genome.  De novo sequencing, on the other hand, is the term used to sequence a genome from scratch. It is much more costly, time-intensive, and limited to select techniques. The length of a read must be at least 1,000 bps long. The first human genome sequenced relied on these methods, which is one of the reasons it was so costly and time-intensive.

"What exactly is NGS?  The textbook definition of Next-Generation Sequencing [also known as massively parallel sequencing (MPS)] is a high-throughput DNA sequencing methodology that makes use of parallelization to process up to half a million sequences concurrently. The process of running thousands of analytes at a time is known as a multiplexing.

"Distinguishing factors of NGS[:] A simpler library preparation * * *

"07. Bridge PCR * * * [Please do NOT view graphic in this section, which is incorrect]

"08. Illumina Sequencing-By-Synthesis (SBS) Technology[:] * * * Illumina uses bridge PCR, which we just saw in the previous page. The sequencing is conducted on a flow cell using sequencing-by-synthesis methods with fluorescent lights. This requires the user of high-resolution optical devices.
History[:] The technology was originally developed by Shankar Balasubramanian[, PhD (Indian-born; his parents moved to UK when he was 1 year old)] and David Klenerman[, PhD] at the University of Cambridge [in summer 1997; they had been doing research by observing DNA polymerase incorporating one deoxynucleotide at a time; that summer, it dawned on them that they can sequence DNA this way; they did not publish paper on this notion because the notion is straightforward; in 2004 they made a critical acquisition (see bullet point)]. The two founded Solexa [based in Chesterford Research Park in Essex (county), close to the Cambridgeshire border] in 1998, commercializing their sequencing method. Illumina [1998- ; based in San Diego, Calif] merged with [acquired] Solexa in 2007 for 600m [not mentioning unit: dollar or pound] * * *
Procedure[:]
1) Library Preparation[:] Whole genomes are fragmented by nebulization or sonication. The randomly fragmented genomic DNA * * *
2) Clonal colony cluster creation[:] The DNA is then placed on a flow cell, which are silica slides of eight lengthwise lengths. These are about the size of a microscope slide, and are sealed to minimize contamination and handling errors.
3) Sequencing [Please view graphic, whose caption reads: 'Sequencing by Synthesis. dNTP fluorescence is translated to a base call.']
* * *
Videos [Please view the first video only, whose title is 'Illumina Sequencing; Overview']


Note:
(a) Latin-English dictionary:
* multiplex (adjective masculine; from adjective masculine multus many + -plex fold [from plexus, which is Latin past participle of plectere to weave)] ):
"1: having many folds;
2: manifold"
https://en.wiktionary.org/wiki/multiplex
(b) Professors had the idea of watching incorporation one a time. But the fluorescence signal of a single deoxynucleotide is too weak, which needs to be amplified. Another  
In 2004 "Solexa acquired Manteias molecular clustering technology. The amplification of single DNA molecules into clusters enhanced the fidelity and accuracy of gene calling, while reducing the cost of the optics on the system through the generation of a stronger signal."  from Illumina archives.

Not Manteias (with an s at the end; likely intended to be Manteia's), but
Manteia Predictive Medicine
https://en.wikipedia.org/wiki/Manteia_Predictive_Medicine
("created in [ ] 2000 as a spin-off of Serono, a Swiss-based biotechnology company * * * The company was basing its strategy on the development of so-called "DNA colony sequencing" technology (now commercialized by Illumina) * * * whose development had been initiated in late 1996 at Glaxo-Welcome's Geneva Biomedical Research Institute (GBRI), by Pascal Mayer and Laurent Farinelli")
(b) Once I realize what is going on in massively parallel sequencing/ Next-Generation Sequencing / Sequencing-By-Synthesis, I am impressed.
(i) A picture is worth a thousand words. So view Figure 2 in
Ballard D et al, Massive Parallel Sequencing in Forensics: Advantages, Issues, Technicalities, and Prospects. International Journal of Legal Medicine, 134: 1291 (2020).
https://link.springer.com/article/10.1007/s00414-020-02294-0

Look at the leftmost column of Fig 2. DNA is randomly fragmented. The beauty is not to sort them (DNA fragment) out or purify them. Instead, one end of each DNA fragment is added an adapter (a short piece of DNA, whose sequence you know and whose exact sequence is not important) and the other end of the same DNA fragment is added another adapter (these two adaptors are represented by two different colors; remember that adaptors are of only two kinds but DNA fragments are all different from one another). The two strands of DNA fragments are separated by heating, for example), where one strand will bind to one adaptor and the other strand will bind to another adaptor (it does not matter which adaptor; see next). The second strand of DNA is then extended (from the adaptor). Then the two strands are separated again and the first strand is washed away. Now the second strand is anchored to the slide (the first has no anchor, and that is why it was washed away.) The second is long, so it bends like a rice sheaf whose adaptor binds to another adaptor nearby. Repeat and yo will obtain a cluster of clonal (identical) DNA fragments. You just watch "Videos" in the quote section above. So each ROUND of base incorporation to a strand of the cluster is recorded. There are different clusters of DNA fragments, which again are all different  in sequences.

Balasubramanian S, Sequencing Nucleic Acids: From Chemistry to Medicine. Cambridge, England: Chemical Communications, 47: 7281 (2011)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428630/
, where you should take notice of "Fig. 3 Solid phase DNA synthesis: and, especially,  Fig. 6 An image of the surface taken during a cycle of an early Solexa sequencing experiment. Each of the spots is a cluster of identical DNA sample fragments and the colour indicates which of the four bases has been incorporated at that particular cycle."  Fig 6 is what the term massively parallel sequencing (MPS) means -- all different DNA fragments are being sequenced at the same time.
(c) Massively parallel sequencing is used in Covid-19 DNA testing.


(2) What is noninvasive prenatal testing (NIPT) and what disorders can it screen for?  Genetics Home Reference, MedlinePlus, National Library of Medicine (NLM), National Institutes of Health, undated
https://medlineplus.gov/genetics/understanding/testing/nipt/
("During pregnancy, the mother’s bloodstream contains a mix of cfDNA that comes from her cells and cells from the placenta [contributed wholly by fetal cells; none from mother]. * * * NIPT is most often used to look for chromosomal disorders that are caused by the presence of an extra or missing copy (aneuploidy) of a chromosome. NIPT primarily looks for Down syndrome (trisomy 21, caused by an extra chromosome 21), trisomy 18 (caused by an extra chromosome 18), trisomy 13 (caused by an extra chromosome 13), and extra or missing copies of the X chromosome and Y chromosome (the sex chromosomes). The accuracy of the test varies by disorder. * * * NIPT is beginning to be used to test for genetic disorders that are caused by changes (variants) [or mutationbs[ in single genes. As technology improves and the cost of genetic testing decreases, researchers expect that NIPT will become available for many more genetic conditions. * * * NIPT is a screening test, which means that it will not give a definitive answer about whether or not a fetus has a genetic condition. The test can only estimate whether the risk of having certain conditions is increased or decreased. * * * Because NIPT analyzes both fetal and maternal cfDNA, the test may detect a genetic condition in the mother. * * * There must be enough fetal cfDNA in the mother’s bloodstream to be able to identify fetal chromosome abnormalities. The proportion of cfDNA in maternal blood that comes from the placenta is known as the fetal fraction. Generally, the fetal fraction must be above 4 percent, which typically occurs around the tenth week of pregnancy. Low fetal fractions can lead to an inability to perform the test or a false negative result. * * * To determine chromosomal aneuploidy, the most common method is to count all cfDNA fragments (both fetal and maternal). If the percentage of cfDNA fragments from each chromosome is as expected, then the fetus has a decreased risk of having a chromosomal condition (negative test result). If the percentage of cfDNA fragments from a particular chromosome is more than expected, then the fetus has an increased likelihood of having a trisomy condition (positive test result). A positive screening result indicates that further testing (called diagnostic testing, because it is used to diagnose a disease) should be performed to confirm the result")

(3)
(a) Terry Taylor, What Is STR Analysis?  National Institute of Justice, Office of Justice Programs, US Department of Justice, Mar 2, 2011.
https://nij.ojp.gov/topics/articles/what-str-analysis
("The most common type of DNA profiling today for criminal cases and other types of forensic uses is called 'STR' (short tandem repeat) analysis. * * * (This analogy fails to capture the complexity of the DNA-protein system, however, because proteins are not only the 'bricks' and 'imber.' Some [proteins, such as those synthesize (during mitosis or meiosis) or repair DNA] 'read' the 'blueprint' and supervise the building, others are the 'bricklayers' and 'carpenters,' [probably meaning the synthesizing of proteins] and still others maintain and keep the house [cell] functioning after it is built.) * * * Given enough Y-STRs, which scientists call loci, a Y-STR profile can offer substantial power to discriminate between individuals, but this type of profile is certainly not as powerful as an autosomal STR profile. * * * In the United States, 13 autosomal STR loci are now accepted as the system used for forensic purposes. Given a robust crime scene DNA sample with good data for all 13 STRs, the likelihood of a person unrelated to the actual perpetrator having a perfect match for all 13 is typically around 1 in 1 billion. By contrast, experimental work with a very robust set of 30 Y-STR loci showed a probability of about 1 in 50,000 for a perfect match") (citations omitted)
(b) STRs are also used in paternity tests.

choi

Oct 18.2021

(1) I noticed when preparing the posting above that each clone /cluster in Manteia Predictive Medicine's Pascal Mayer and Laurent Farinelli has two kinds )proportion being 50/50) of single strand, corresponding to the two strands of a double-stranded DNA. During the weekend after publication of the blog, I was haunted by how one strand is removed from the cluster.
(2) Just now, I re-read Figure 2 in
Ballard D et al, Massive Parallel Sequencing in Forensics: Advantages, Issues, Technicalities, and Prospects. International Journal of Legal Medicine, 134: 1291 (2020).
https://link.springer.com/article/10.1007/s00414-020-02294-0
, whose caption simply stated, "Subsequently, the 3′ block and the fluorescent tag on the incorporated nucleotide are removed." But how was "3' block" (or strands) removed (as they were anchored, too)?  ("[T]he fluorescent tag on the incorporated nucleotide" can be removed by somehow cleaving it from the last deoxynucleotide incorporated.) Text in this Webpage did not explain, either.

Removing one strand in a cluster is crucial. Otherwise a cluster  would not be a clone.
(3) Method of Nucleic Acid Amplification. Patent number: 9902951; Date of Patent: Feb 27, 2018' Assignees: Illumina, Inc and Illumina Cambridge Limited (in UK, not Cambridge, Massachusetts); Inventors: Eric H. Kawashima, Laurent Farinelli, Pascal Mayer.
https://patents.justia.com/patent/9902951  
("In another embodiment the colonies can be partially digested with a double-strandspecific [sic: should be 'double-strand specific'] 3' to 5' DNA exonuclease (see section E, FIG. 12C, (f)) which removes one strand of DNA duplexes starting from the 3' end, thus leaving a part of a DNA molecule in single stranded form. Alternatively,DNA in colonies can first be heat denatured and then partially digested with an single-strand specific 3' to 5' DNA exonuclease which digests single stranded DNA starting from the 3' end")

There are two types of enzymes that cut DNA (nucleases), exonuclease and endonuclease.

"Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules [double-stranded DNAs]."  en.wikipedia.org for en.wikipedia.

There are many exonucleases that remove or eat away a nucleotide one at a time from the end(s) (5' or 3' or both). Nad There are many others who do the same on RNA.