Next-generation sequencing (NGS) is a high-throughput technique that allows millions of nucleic acids to be sequenced simultaneously in parallel.


Key features


It enables high-throughput and cost-effective sequencing, facilitating clinical use.

Basically different methods of next generation sequencing short read (pirosekanslam, synthesis with sequencing, ligation sequencing with Ion Torrent) and long-read (Single molecule real time sequencing, sequencing nanogozenek) are divided into two.

Clinical applications primarily include oncology panels and germline panels, along with emerging whole exome sequencing/whole genome sequencing.


Terminology


Next-generation sequencing (NGS), massively parallel sequencing, whole genome sequencing (WGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), methylation sequencing (methyl-seq), and chromatin immunoprecipitation sequencing (ChIP-seq)


History

In the early 20th century, many studies attempted to elucidate cell signaling pathways and cellular building blocks. Biomark Insights 2010;5:9 , Int J Mol Sci 2019;20:3292 , Nature 2017;550:345 )

  • 1921 – 1922: Frederick Banting and Charles Best discovered the peptide hormone insulin.
  • 1953: Fred Sanger published the amino acid sequences of the amino acid sequence of insulin and other proteins has initiated efforts to determine.


Success in determining the amino acid sequences of proteins inspired efforts to sequence genetic material ( Nature 2017;550:345 )

  • 1965: The first RNA sequences were determined and published (the first sequence identified was alanine tRNA)
  • DNA sequencing proved more challenging and laborious until Sanger and Coulson and Maxam and Gilbert published higher-throughput DNA sequencing methods in 1976–1977.
    • Sanger and Coulson method: chain termination using DNA primers and DNA polymerase
    • Maxam and Gilbert method: restriction fragmentation and base-specific chemical cleavage

Both the methods of Sanger and Coulson (Sanger sequencing) and Maxam and Gilbert were immediately used and spurred the exponential growth of new DNA sequencing projects ( Nature 2017;550:345 )

  • Shortly after both DNA sequencing methods were published, shotgun sequencing methods (sequencing random clones with combinations determined by overlapping sequences) were published in 1979–1980.

The growth of DNA sequencing efforts has heralded advances in bioinformatics technology and resources, including databases and search tools (e.g., NCBI BLAST, GenBank, etc.) Nature 2017;550:345 )

  • 1980s – 1990s: massively parallel sequencing methods have been described
  • 1990 – 2004: The Human Genome Project, considered the most ambitious sequencing project undertaken at the time, was carried out and the final sequence of a large portion of the human genome was published in 2004.
    • The ambitious nature of the project led to advances in bioinformatics and genetic technologies (requiring more efficient processes with higher yields and accuracy, etc.)
  • 2000s – 2010: Massively parallel sequencing methods replaced Sanger sequencing methods and the first NGS machines became commercially available.

Sanger sequencing


gold standard

  • The historical gold standard for providing high accuracy in long reads; however, the accuracy (99.965% confirmation rate), efficiency, and cost-effectiveness of NGS methods have largely eclipsed Sanger sequencing in clinical and research settings ( Clin Chem 2016;62:647 )


Method: It uses chain termination with modified dideoxynucleotides to determine the sequence.


Advantages: Can sequence any target (DNA, RNA, epigenetic modifications), high quality, long read lengths.


Disadvantages: high cost (especially for longer sequences and when sequencing multiple genes/loci), low throughput, time consuming and insufficient sensitivity to identify somatic variants and structural variants in tumor samples, requiring knowledge of the sequences of interest for primer design and amplification.


Use: Validation tool for NGS data; its use as a primary sequencing method in clinical and research settings is declining.


Overview of NGS methodology


It uses massively parallel sequencing methods consisting of library preparation, amplification, and sequencing.


Library (sample) preparation: Random fragmentation of DNA followed by ligation of common adaptor sequences

  • Sample input: DNA molecules (from blood, bone marrow, cheek swab, saliva, formalin-fixed paraffin-embedded tissue, etc.)
  • Fragmentation: Fragment DNA enzymatically (restriction enzyme, transposase), sonically, or mechanically to random sizes (usually 200–300 nucleotides for short-read sequencing)
  • Modification of target DNA
    • Adapters: Unique DNA sequences that serve multiple functions are added to the ends of fragmented DNA samples
      • Provide a barcode (index) containing patient identifiers for multiple processing of different samples in the same study
      • Allow hybridization of arrays to sequencing chips/beads
      • Serve as universal priming points for amplification and sequencing primers
  • Enrichment: This step is only necessary if specific genomic regions (disease gene panels, exomes, etc.) are being analyzed.
    • PCR amplification (amplicon)
      • Advantages: ideal for smaller genomic regions
      • Disadvantages: May miss target of interest (lower precision)
    • Sequence capture/hybridization
      • Baits (biotinylated RNA or DNA oligonucleotides) bind to specific regions of DNA
      • Advantages: ideal for larger genomic regions
      • Disadvantages: lower enrichment for target regions due to off-target capture (lower specificity)


Creating a cluster amplification and

  • Each molecule from a single piece of template-derived PCR multiplex PCR by using aggregation, which enables amplikon of the original molecule in close proximity to a surface (chip flow cells, beads, nano-sphere, etc.) and immobilized on replicated ( Nat Rev Genet 2016;17:333 )
    • Bead-based systems: a specific oligonucleotide complementary to the adapter and a bead secured millions clonal fragment 1 bead emulsion PCR amplification of the DNA fragment takes place through a single fixed on where they are
    • Solid surface systems: constants in a specific location on the chip adapter oligonucleotides
      • PCR amplification takes place via the hyperlink on the slide directly; forward and Reverse Primers binds to the surface here, and this complementary single-stranded DNA fragments provides binding sites for
      • Flow cells on the slide system identifies the exact location of the primers patterned surface which provides higher efficiency and
    • DNA nanoball: in the solution, the DNA binds again and again, and is cut by a unique 4 zone dairesellestirili adapter
    • Then, slide a patterned distributed on DNA nano-spheres rolling circle amplification is used to create


Sequencing

  • Each cluster will act as a separate sequencing reaction, and at the same time from multiple samples in parallel libraries can dizileneb
  • Reading: sequencing output; the sequence of the nucleotide sequence representing the original template molecule
  • The couple has three readings: readings from both ends of the DNA template and both forward and back dizilenir advanced mapping, coverage and allows for efficiency ( Nat Rev Genet 2016;17:333 )
    • Reading back and forth between asymmetry exists, may help to identify the structural arrangements
  • NGS platforms to be categorized ( for information about the methods and functions of the platform Refer to the section NGS platforms for)
    • Generations ( Nat Rev Genet 2016;17:333 )
      • Second generation: PCR-dependent
        • Pirosekanslam (the Roche 454 system)
        • Ligation with sequencing (AB SOLiD system, Complete Genomics, Polonator G. 007, etc.)
        • Through synthesis sequencing (Illumina, Qiagen GeneReader)
        • Ion semiconductor sequencing (Ion Torrent, Thermo Fisher)
      • Third generation: PCR without the need for real-time sequencing
        • Single molecule real time (SMRT) (PacBio, Roche)
        • Nanopore (Oxford Nanopore Technologies)
  • A long read and short read ( Int J Mol Sci 2017;18:E308 )
    • Short reading:
      • Examples: a synthesis through sequencing, ligation through sequencing, to pirosekanslam
      • Advantages: low cost per Gb, high accuracy
      • Disadvantages: Reduced alignment, identification and resolution of structural arrangements limited
    • Long read: 2,5 KB is larger than read only.
      • Examples: single-molecule real time (SMRT), nanopore
      • Advantages: improved alignment, structural variations, and the detection of a major reorganisation, repetitive regions dizilenme new discovery of the RNA transcript isoforms, the processing time PCR improves portability and not to be trusted.
      • Disadvantages: high cost per GB for low accuracy
        • Bioinformatics methods and optimization algorithm to increase the depth of reading, including the cost of a method, although it is still short more reading accuracy up to 98% has increased ( Nat Rev Genet 2020;21:597 )
Fixed with formalin and paraffin embedded tissue (FFPE)

The most common example is one of the types used for genetic testing. Not necessarily the pathology laboratory by a pathologist at the sample should be selected. Free samples of tumor tissue enough false-positive or false-negative results may result. For example, tumor tissue contains enough of it-it means that it contains. The pathologist who examined under a microscope to decide. FFPE NGS testing provides clinically appropriate for an example of the type.

General workflow

  • Tru-cut biopsies and fine needle aspiration (Fna) is often used in order to obtain tissue for the diagnosis of the primary tumor.
    • Excisional biopsies and surgical resection specimens, such as larger, less frequently for the initial diagnosis are obtained.
  • Tissue fixed with formalin and embedded into paraffin blocks for histological evaluation are.
  • Sectioned tissue for diagnosis is evaluated in terms of morphology or immunofenotip.
  • The remaining tissue in the paraffin block, including molecular analysis with NGS can be used to help test.

With this workflow, a new diagnostic tests necessary for the diagnosis of cancer, only a small biopsy material obtained from the patient and thus minimize exposure to more invasive surgical procedures can be completed on when the clinician to optimize the management of therapeutic help.

The advantages of NGS tests for FFPE

  • Archived tissue
    • FFPE tissue DNA is relatively stable and allows for retrospective analysis of tumors.
    • The quantity and viability of DNA decreases with time at ambient conditions (in some studies, the amount under 50% has dropped, and 4-6 years after only 11% can be replicated) ( 2022 J Pers Med;12:750 )
    • FFPE tissue FFPE store or freeze at lower temperatures, has been proposed as a way to better protect the genetic material in FFPE (however, there is a need for more data to evaluate this proposal will increase the cost of the store and this is probably FFPE) ( 2022 J Pers Med;12:750 )
  • Histological analysis
    • Diagnosis in addition to the morphological analysis of the samples, NGS in the last instance, in order to maximize the percentage of the tumor to non-tumor sample of the tumor from the rest of makrodiseksiyon allows.
    • Some new research by applying NGS tumor immunohistochemical profile with different genomic regions can be able to distinguish the differences between regions with different immunofenotip indicate that the tumor; however, additional process steps that need to immunboya in some studies has been shown to decrease the amount and quality of the DNA of ( PLOS ONE 2017;12:e0176280 )
  • Ease of carrying
    • Cut back on FFPE tissue blocks or slides, portable and does not create significant biological hazards at ambient conditions (fresh tissue, blood, bone marrow, cerebrospinal fluid, etc. the processing of such samples as compared to more stringent handling requirements associated with the risk of infection and potential ().

Challenges associated with obtaining the Ideal FFPE samples

  • Before analysis, reduction of errors
    • The samples as soon as possible to reduce hot and cold ischemic time should be placed into formalin and taken from the patient (with the appropriate sections of formalin to enable them to penetrate more evenly).
      • Prolonged warm ischemia (when the sample is removed from the patient, with the decrease of the perfusion occurs) and cold ischemia (formalin specimen placed in the/during storage occurs) increases and reductions, including transcripts of genetic material in a number of potential degradation and specific genetic changes associated with RNA, DNA because it is more unstable compared to the potential are especially prone to the effects of deterioration) ( 2022 J Pers Med;12:750 )
    • During sampling, the percentage of tumor volume and tumor should be optimized.
      • The amount of sample available for analysis of tumor NGS that have a crucial influence on the success of the test, pre-analytical factors ( Am J Clin Pathol 2016;145:222 )
      • Typically, the sample is sent to test at least 20% of tumor is recommended Am J Clin Pathol 2016;145:222 )
      • Many manufacturers NGS to achieve the greatest success in the Test at least 10 ng recommends the removal of tumor DNA ( Am J Clin Pathol 2016;145:222 )
        • Platforms such as Illumina, Hybrid Capture test for at least 15,000 recommends tumor cells per sample ( 2022 J Pers Med;12:750 )
        • Ion Torrent platform, per experiment, 100 – 1000 tumor cells requires 2022 J Pers Med;12:750 )
      • Larger samples (Excision and resection of the tumor samples compared to FNA and core biopsy to obtain a larger percentage of tumor volume and is more likely.
      • Tru-cut biopsies and fine needle aspiration biopsy from the tumor insufficient parts/tumor cell clusters may or non-tumor cells (e.g., inflammatory cells and the surrounding normal tissue compared to tumor cells may result in samples that contain a low proportion.
        • Hucresellig low tumor, tumor cells can interfere with specific genetic analyses to be meaningful.
      • Tru-cut biopsies and fine-needle aspiration biopsy samples, such as smaller or more limited samples is achieved when the biopsy material of rapid onsite evaluation (ROSE), such as processes, and help both morphological tests (NGS analysis, such as in the last example in order to maximize the volume of the tumor can help in the classification of sample ( 2022 J Pers Med;12:750 )
  • Formalin fixation of optimum quality and quantity sufficient to provide genetic material for NGS studies is critical.
    • According to morphology and ancillary studies such as NGS for the analysis of samples with 10% formalin in 12 – 24 hours is recommended fixation ( 2022 J Pers Med;12:750 , Int J Mol Sci 2016;17:1579 , PLOS ONE 2017;12:e0176280 )
    • Inadequate fixation, enzymatic degradation of tissue and genetic material may cause.
    • Excessive fixation, formalin fixation of works depending on more (some modification up to 1 500) can lead to the formation of.
    • During the creation of NGS libraries and removing the genetic material, DNA cross-links and take out some of uracil and high-volume to reduce additional steps, such as enzymatic processes ssDNA fragments, fixation artifacts and CNV and single nucleotide variants (SNV) can help reduce false detection.
  • If possible, additional tissue processing steps (e.g. removal of calcification) should be skipped.
    • The success of NGS became significantly lower bone and other calcified textures associated with it. ( 2022 J Pers Med;12:750 , Am J Clin Pathol 2016;145:222 )
    • Lime solving (dekalsifkasyon), destroys the genetic material that a strong (hydrochloric and nitric) or weak (picric, formin, acetic) acid or builder chelating agents (EDTA) requires the use of.
      • Short-term exposure to weak acids and EDTA, SNV of Fusion transcripts is still sufficient for the detection of CNV and may allow the extraction of DNA. ( 2022 J Pers Med;12:750 )
    • In general, a biopsy of the primary tumor has spread to the bones and other hard tissues and a large number of if a biopsy to be taken off the bone metastasis of NGS through the tissues to be analyzed in a successful manner are more likely to be in obtaining that may result. ( Am J Clin Pathol 2016;145:222 )
  • Removing the genetic material from FFPE methods, can significantly affect the amount and quality of material that is used for NGS.
    • Studies of different extraction kits (columns, magnetic beads, etc. as such, different purification methods using reagents or) the final yield of the extracted material, has shown that differ in terms of purity and quality. ( Int J Mol Sci 2016;17:1579 )
    • Especially in low tumor volume/percentage when working with small samples, the efficiency of the extraction process, it is essential to maximize quality and productivity. ( Int J Mol Sci 2016;17:1579 )
Data output and analysis

Demultipleksleme: Unified dizilenen simultaneously in patient samples (sample library) then each patient sample-specific barcodes (with indexes) is divided into

Readingssequence similarity based on readings are clustered and back and forth physically aligned (mapped)

Alignment

  • Re-sequencing: Array read array reference (the reference genome of an individual) is aligned according to.
  • The de novo sequences to each other and aligned in a manner that is read.

Comments: pathogenic and possibly pathogenic, variant of uncertain significance, probably benign, benign ( J Mol Diagn 2017;19:4 , Genet Med 2015;17:405 )

NGS performs the advantages of (compared to Sanger sequencing)
  • High efficiency/output
  • Improved resolution
  • Cost-effective
  • There are algorithms that can detect structural differences
NGS performs the disadvantages (compared to Sanger sequencing)
  • Shorter read lengths
  • In the areas of Homology (psodogen like), again in the expansion, large insertions, copy number variants (CNV), or other structural variants in the decrease in accuracy raw
    • Genetic events are usually non-unique sequences in centromeric DNA repetitive elements, etc.) NGS occur and this makes it difficult to identify with
  • High startup costs
Platforms for NGS

To pirosekanslam

  • The first commercially successful NGS System
  • Sequencing mechanism
    • Beads template linked with the enzyme-containing beads on a plate containing distributed.
    • 4 to 1 or more nucleotides from each of the sequencing reaction is added as iterative.
    • Participation during nucleotide pyrophosphate (PPI) the PPI is released and the release of participating is equal to the amount of the nucleotide.
  • Example: 454 system (Roche)
  • Advantages: fast, long read length
  • Disadvantages: the high cost of the reagents, polibase > 6 with a relatively high error rate, low yield ( J BioMed Biotechnol 2012;2012:251364 )

Ligation with sequencing

  • Sequencing mechanisms: Labeled probe hybridization to the DNA strand and connecting
    • 2 specific probe oligonucleotide probes containing base, and 6 base dejenerat uses oktamer
    • Each probe some last-attached fluorescent dyes, one of the first to fifth on the basis of the field and 4 ligation on the basis of separation will have the space
    • Sequencing occurs between the probe and the complementary connecting with the template, and Anchorage known as complementary to adapter ligation serves as a launching point for the blind
    • Fluorescence is displayed later freed
    • The fluorescent signal and the last 4 nucleotides are removed and the cycle continues oktamer
    • A couple of hybridization, ligation and cutting cycles after sequencing some DNA is denatured and the reaction is used as primers to repeat another wrapped with 1
    • Sequencing the Sequencing Primers used a total of 5 about the sequence of Round 5 can be removed after
  • Example: EU-solid system (Thermo Fisher)
  • Advantages: the second generation of NGS performs the highest accuracy, be dependent on the polymerase
  • Disadvantages: short-read sequencing ( Nat Rev Genet 2016;17:333 , J BioMed Biotechnol 2012;2012:251364 )

Sequencing through synthesis (SBS)

  • One of the methods most commonly used in clinical practice ( Arch Pathol Lab Med 2017;141:1544 )
  • Sequencing mechanism
    • In each cycle, different fluorescent dyes in the flow cell that contains all 4 detachable and removable blocking group deoksinukleotid triphosphate (dNTP) is added to the
    • By the inclusion marked with fluorescent DNA polymerase from each polymerization cycle terminates, and the fluorescent signal, determines specific nucleotides on the DNA strand growing
    • The blocking group is insignificant as enzymatic and fluorescent dye, thus a new nucleotide can be added during the next cycle
  • Example: Illumina sequencing (MiSeq, HiSeq, and NovaSeq NextSeq platforms)
  • Advantages: high accuracy (base sequences), the largest output, best inexpensive, single nucleotide insertion platforms (Ion Torrent, pirosekanslam) according to the homopolymer is less sensitive to errors
  • Disadvantages: the circular structure of the long-time sequencing, and combine short reading leads to Coleman: Diagnostic Molecular Pathology, 2016 , Nat Rev Genet 2016;17:333 , J BioMed Biotechnol 2012;2012:251364 )

Ion semiconductor sequencing

  • One of the methods most commonly used in clinical practice ( Arch Pathol Lab Med 2017;141:1544 )
  • The sequencing mechanism: Ion Torrent sequencing (semiconductor sequencing)
    • Enriched DNA fragments on the beads, and then a micro-well plates are arranged into so that only one bead occupies a well
    • Mikroku unmodified nucleotides are added sequentially to a single chip (with the addition of single nucleotide sequencing)
    • The inclusion of complementary nucleotides by DNA polymerase nucleotides detected by an ion sensitive to changes in PH when added to each of the sensor causes the release of a proton
    • In addition, the evaluation of each successive nucleotide is determined by the intensity of the electrical signal during exposure
  • Example: Ion Torrent (Thermo Fisher)
  • Advantages: fast sequencing (camera dependent fluorescent or if not), lower cost, smaller device size
  • Point-of-care tests and is useful for gene panels
  • Disadvantages: increased accuracy with error less homopolymer ( Nat Rev Genet 2016;17:333 )

Single molecule real time (SMRT) Sequencing

  • In comparison with other systems, clonal amplification of the target region for each dNTP is added unreliable or chemical loop
  • Sequencing mechanism
    • Polymerase immobilized target molecule, and each one containing a copy of the thousands of micro-wells with a special flow cell, a circular template that can usually multiple times dizileneb
    • Mikrokuyu, is filled with more marked with fluorescent nucleotides
    • Paint, to be incorporated by DNA polymerase during disintegrate, painted and DNA polymerase dNTP away from the sensor Active Region will enable us to make available for the next
    • Mikrokuyu the light detector, nucleotides, as it detects the signal in real time
  • Example: Pacific Biosciences
  • Advantages: the fastest method, longer sort the targets, PCR attributed reduces prejudice and error, the new genome is ideal for applications that merge real-time production data
  • Disadvantages: accuracy and a lower yield ( Nat Rev Genet 2016;17:333 , J BioMed Biotechnol 2012;2012:251364 )
    • Platform performance improvements, increased yield (PacBio Sequence II 160 GB per flow cell on the platform, including increasing read depth (coverage genomic 40 times that it lets me, and as a result the accuracy of the consensus between the readings of 99.9% has increased ( Nat Rev Genet 2020;21:597 )
    • High accuracy readings (those that are generated by the PacBio platform, such as more time and resources (including computational processing power) and requires more than one molecule per cell due to the production of each template and sequencing genomic merge results in a higher cost per reading, however, produced readings and longer (> 10 Kb) is true for both (>99%) ( Nat Rev Genet 2020;21:597 )

Nanogozenek

  • Compared with other systems, this system will monitor the inclusion of nucleotides or a secondary signal (fluorescence, pH, etc.) it does not use.
  • Sequencing mechanism
    • The ionic current Nano-pore proteins is passed from
    • The target DNA is then a nano-pores is passed from each base flow changes
    • The DNA molecule nano-pores passing takes place in real time sequencing
    • The hairpin structure of the threads library allows you to dizilenme back and forth (for example, Oxford Nanopore Technologies)
  • Advantages: very long molecules. sort (standard length: 10 kb to 100 KB; ultra-long readings: > 100 Kb), lower cost, small and portable, real-time data production; de novo assembly, structural variants ( Nat Rev Genet 2020;21:597 )
    • The flow cell per platform), the result (for example, Oxford Nanopore Technologies capable of running at the same time the flow cell 48’ PromethION), some as short as yield potential Reading-based platforms (e.g., Illumina NovaSeq) may exceed the yield ( Nat Rev Genet 2020;21:597 )
  • Disadvantages: relatively high error rate ( Nat Rev Genet 2016;17:333 , J BioMed Biotechnol 2012;2012:251364 )
    • The accuracy of the basic calls are to a large extent depends on the algorithm used; these algorithms, improvements in the standard readings (10 – 100 kb), the average accuracy of 87% – 98% has increased, the consensus reading is the accuracy of 97% – 98%) is close to ( Nat Rev Genet 2020;21:597 )

Computer Science


Overview: Raw sequencing data into separate arrays (readings) are translated to when a targeted region of the genome are mapped to each reading, and then determines the differences between this alignment with standardized reference DNA sample.


Base call and demultipleksleme: Raw data (fluorescence intensity, electrical stimulation, etc.) target nucleotide in the DNA sequence is converted to a sequence that is assigned to each position (e.g., binary base call (BCL) format)

  • According to the background noise signal density, basic call allow you to create a confidence score associated with each nucleotide of the algorithm
  • The nucleotide sequence containing the read data read from identifier BCL converted to FASTQ format and confidence scores demultiplekslenir
    • FASTQ format:
      • Line 1: @Sequence identifier (and optional description)
      • Row 2: a raw array would be read as: “ACTGACTG”
      • Row 3: Space: ‘+’
      • Row 4: Row 2 of Phred quality score (probability sequencer has been called by the base in a wrong way): ‘!AA>**!C
      • S = -log(10)P
      • Q = Phred quality score
      • P = base the possibility of a false call (calculated with the peak shape and overlap in the bases)
      • Sample software: CASAVA (Illumina)

Sequence alignment:

  • All readings may be mapped to the locations on the genome of the individual
  • The read data and the array reference (where a standard is aligned readings and comparison of individual needs
    • Biological factors (benign variants and mutations) and technical factors (sequencing errors or incorrect base calls) contributes to the imperfect alignment
    • The penalties for incorrect alignment is produced; on the verge of a specific penalty, a read is considered to be align
  • Scope (depth): aligned on a single base of a nucleotide or read the number of the number of time dizilendi
    • Enhanced coverage in that location are represented correctly validates the basic call sequence and basic increases confidence that the original target of the call
    • From some of the germ line of pure, or twenty times to make sure the coverage mentioned in the example (20 reading over the target area, usually specified as 20x) is required
    • Mixed samples such as tumor variants in the samples to determine a thousand times to five hundred (500 – 1.000 x) are needed to cover
  • Alignment formats: sequence alignment/map (Sam) and binary Bam format alignment reference in the array A stores the data according to the position check box
  • Sample software: BWA, Bowtie 2, MAQ, Stampy, Novoalign

Variant call:

  • Are aligned to the reference genome once, single nucleotide polymorphisms (SNPs) and single nucleotide variants (SNV) and insertion/deletions (indel) can be defined in
  • Scope, depth, variable frequency, and the alignment score considers
  • File format: variant call format (VCF), mutation description format (MAF)
  • Sample software: Sam tools Mpileup, MTM

Visualizing data:

Reference: Arch Pathol Lab Med 2017;141:1544 , An Rev Pathol 2020;15:97


NGS applications

DNA sequencing

  • Data analysis: point mutations, insertions/deletions (indel), and copy number variants (CNV), structural variants; the tumor mutation burden (TMB), determines the signature mutation
  • Whole genome sequence
    • Both of the genome that encode both non-coding regions of the sequences
    • Advantages: does not require amplification or enrichment Library preparation (high specificity)
    • Disadvantages: high cost, less depth coverage, analysis and interpretation of complex data
  • All ekzom sequence
    • Only protein-coding the field of dizilenme of the genome (~%1 – 2)
    • Mutation per nucleotide specificity and sufficient sensitivity for detection of > 20x coverage is needed
    • Advantages: it is more economical for clinical use
  • Targeted sequencing
    • Uses for specific regions of the genome with sequence specific primers and enriching
    • Advantages: it allows the execution of a greater number of individual sample in each sequencing reaction, reduces costs and increases the depth of the region of interest kapsanma
  • Gene panels
    • Genomic medicine
      • Hereditary syndromes (eg, Lynch syndrome, hereditary breast/ovarian cancer syndrome) or a mitochondrial diseases
      • 80x depth is sufficient to identify germ line variants
    • Somatic mutations
      • Hematolenfoid neoplasm and solid tumor panels
      • >There is a need for somatic mutations to cover 500x

RNA sequencing (RNA-seq)

  • Reverse transcriptase PCR (RT-PCR) uses
  • Data analysis: different expression, gene fusion, RNA editing and alternative insert

Other

  • Methylation sequencing (methyl-seq): to analyze DNA methylation patterns with DNA uses processed bisulfit
    • It is useful for analysis of epigenetic changes, such as parental pressure
    • The methylation profile, CNS tumor classification is becoming increasingly important for ( 2021 Neuro Christoph F;23:1231 )
  • Immunocokturme chromatin (ChIP-seq): immunocokturme Chromatin DNA-protein interactions using analysis of Coleman: Diagnostic Molecular Pathology, 2016 , Genet Med 2013;15:733 )
  • Transpozaza accessible chromatin with sequencing for the experiment (paper clip-seq)
  • Cut and run

Clinical applications


Oncology tests


Usually hematologic malignancies or solid-specific panels to identify specific gene variants or somatic sequence variant uses.

  • Helps in diagnosis
  • Provides prognostic information
  • Effective treatment options (targeted therapies)
  • BRAF V600E mutation of the BRAF inhibitor (vemurafenib for example are treated with

Commercial tests (for example FoundationOne) allows for the testing of the genomic region, hundreds of

Germ line test

To identify germ line DNA variants WES, WGS, targeted germ-line or used in panels.

  • Diagnose hereditary diseases
  • Explain the phenotypes that are probably genetic in origin
  • Cancer susceptibility ( BRCA1 / BRCA2 mutations or REQUIRED, etc.) determine

SNP detection and analysis allows commercial tests (e.g. 23andme Ancestry, Gene Dx, Ambry Genetics, etc.)

Microbiology

Metagenomik and WGS sequencing methods are used for the following purposes:

  • Pathogens and mechanisms of therapeutic resistance
  • Monitoring antimicrobial resistance both inside and outside of healthcare organizations
  • The epidemiological monitoring of pathogenic strains of pathogens and disease outbreaks through genotiplendirilm ( Clin Microbiol Infect 2018;24:335 , Genoa (Basel) 2022;13:1566 )

Pharmacogenomics

Genomic variations in drug response or drug-drug interactions in phenotypic differences connect ( Cold Spring Harb Perspect Med 2019;9:a033027 )

Emerging diagnostics

Single-cell transcriptomics, spatial transcriptomics, single-celled array PAPERCLIP

The optical genome mapping (OGM)

  • First introduced in 1990 genome imaging techniques are based on
  • Long linearized DNA molecules (single-stranded piece length of 0.15 – 2.5 MB ranging from high molecular weight DNA derived from) sequence motifs with specific fluorescent markers (usually about 15 to 100 KB per label) are labeled Nano-pore is passed through the channels and high-throughput imaging is analyzed through.
  • In comparison with a reference genome is detected then the pattern of the fluorescent tag is used to create a new genome
  • According to the reference genome map label in the pattern variations, additions, deletions, in reverse, including displacements and are used to detect chromosomal structural variations aneuploid
  • Benefits
    • NGS and comparative genomic hybridization arrays (aCGH), a number of genetic variants-copy number variation according to the level and can detect while, OGM balanced structural variants (e.g. balanced translocations and reverse) and a high resolution variant of the turning points may have better identify and complex structural rearrangements.
    • Repetitive regions of chromosomes, which are historically difficult to dizilenme more easily mappable
      • Many genetic event of non-unique/as occurs in repetitive sequences, OGM, aCGH, and NGS structural arrangements in these areas has the potential to detect what can be done with currently more effectively
    • Insertions and deletions can be detected as small as 500 bp
    • The creation of the OGM data, compared to traditional cell culture and can have a shorter turnaround karyotiplemey, and this allows you to quickly identify potentially more structural variants
  • Limitations
    • Structural and genetic variants must be large enough to be resolved or can be determined by
    • Some regions of the chromosome mapping is not good (for example, some studies subtelomerik after resolution of the X chromosome structural rearrangements of the region reveals the weak to not be able to detect specific)
    • OGM analysis can identify structural variants per sample, more than one and sometimes dozens or hundreds (or more) may result in the identification of potential variants.
      • Considering the relative newness of technology research and potential clinical environments, existing data are limited to filtering settings and strategies
      • Analytical algorithms for copy number neutral loss of heterozigotlug is missing
    • The reference Current is derived from a relatively small cohort maps
    • The current scaling capabilities in terms of clinical applications is limited.

Overall, OGM, scale-up capabilities and analytical advances in the improvement of algorithms, waiting for diagnostic tests (whether obtained from the primary study be like other traditional diagnostic test for verification) promises to make a useful contribution to ( Am J Hum Genet 2021;108:1423 , Genoa (Basel) 2021;12:1958 ).