Next generation sequencing in diagnostic

Metode sekvenciranja novih generacija su najznačajnije otkriće u znanosti biologije u proteklih 40 godina. Nakon Sangerove metode koju nazivamo metodom sekvenciranja prve generacije, razvijaju se metode druge i treće generacije sekvenciranja. Metode sekvenciranja novih generacija daju rezultate u sve kraćem vremenskom periodu te su svakim danom sve učinkovitije. Danas je moguće sekvencirati tisuće i tisuće genoma spomenutim metodama, što omogućava otvaranje mnogih puteva u raznim područjima znanosti i uvelike pridonosi kvaliteti života. Ove metode su veoma značajne u području dijagnostike, tako što omogućuju brže i učinkovitije otkriće raznih kromosomskih nepravilnosti koje dovode do nasljednih bolesti, čestih bolesti u nekoj populaciji te malignih tumora. U ovom radu izložen je kratak povijesni pregled razvoja raznih metoda sekvenciranja kroz zadnja desetljeća , njihova uporaba u raznim područjima, ponajviše njihov doprinos u kliničkim laboratorijima i predstavljanje tih metoda kao novog alata u otkrivanju uzroka mnogih bolesti.New generation-sequencing methods are the most important discovery in biology in the past 40 years. After Sanger sequencing, which is also called the first-generation sequencing, the second and the third-generation sequencing are developed. The new-generation sequencing methods are showing results in shorter time periods and are getting more efficient day by day. Today it is possible to sequence thousands and thousands of genoms with these methods, and this opens many pathways in many areas of science and contribute to the quality lives. These methods are highly significant in many fields of diagnostics, because they help to determine chromosomes irregularities, which cause hereditary diseases, common diseases in a population, and malignant tumors faster and more efficient. This work presents a short historical overview of the development of various sequencing methods through the last decade , their use in various fields , particularly their contribution to clinical laboratories and presentation of these methods as a new tool in detecting causes of many diseases

Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers

BACKGROUND: Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to gamma delta T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. METHODS: The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in gamma delta T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. RESULTS: The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of gamma delta T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. CONCLUSIONS: Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.Peer reviewe

Next generation sequencing in diagnostic

Metode sekvenciranja novih generacija su najznačajnije otkriće u znanosti biologije u proteklih 40 godina. Nakon Sangerove metode koju nazivamo metodom sekvenciranja prve generacije, razvijaju se metode druge i treće generacije sekvenciranja. Metode sekvenciranja novih generacija daju rezultate u sve kraćem vremenskom periodu te su svakim danom sve učinkovitije. Danas je moguće sekvencirati tisuće i tisuće genoma spomenutim metodama, što omogućava otvaranje mnogih puteva u raznim područjima znanosti i uvelike pridonosi kvaliteti života. Ove metode su veoma značajne u području dijagnostike, tako što omogućuju brže i učinkovitije otkriće raznih kromosomskih nepravilnosti koje dovode do nasljednih bolesti, čestih bolesti u nekoj populaciji te malignih tumora. U ovom radu izložen je kratak povijesni pregled razvoja raznih metoda sekvenciranja kroz zadnja desetljeća , njihova uporaba u raznim područjima, ponajviše njihov doprinos u kliničkim laboratorijima i predstavljanje tih metoda kao novog alata u otkrivanju uzroka mnogih bolesti.New generation-sequencing methods are the most important discovery in biology in the past 40 years. After Sanger sequencing, which is also called the first-generation sequencing, the second and the third-generation sequencing are developed. The new-generation sequencing methods are showing results in shorter time periods and are getting more efficient day by day. Today it is possible to sequence thousands and thousands of genoms with these methods, and this opens many pathways in many areas of science and contribute to the quality lives. These methods are highly significant in many fields of diagnostics, because they help to determine chromosomes irregularities, which cause hereditary diseases, common diseases in a population, and malignant tumors faster and more efficient. This work presents a short historical overview of the development of various sequencing methods through the last decade , their use in various fields , particularly their contribution to clinical laboratories and presentation of these methods as a new tool in detecting causes of many diseases

Stargardt disease-associated in-frame ABCA4 exon 17 skipping results in significant ABCA4 function

Abstract Background ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping. Methods A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids. Results Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids. Conclusions Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients. Graphical abstrac