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Illumina sequencing is performed using sequencing-by-synthesis on a flow cell. Short-read sequencing generates — base pair lengths called reads. The reads go through quality control and are aligned to a reference genome before being evaluated for significant characteristics. The method chosen for a given experiment depends largely on what you hope to learn from the experiment. NGS Applications Whole Genome Sequencing is used when comparing genotypes and a comprehensive evaluation of the genome is needed, such as when researching rare diseases.
Whole exome sequencing is a fast and cost-effective approach to interrogating protein-coding genes in a genome and is often used for tumor normal sequencing. Each type of NGS method comes with its own unique benefits and challenges. Talk to a scientific application specialist to choose the right one for you and your experiment. This extensive application guide provides chapters on NGS workflow, as well as types of sequencing and applications.
Unlike other techniques, it does not use fluorescence and does not use modified nucleotides or optics. Instead, pH changes are detected by semiconductor sensor chips and converted to digital information. During the synthesis reactions, the fragments bind to oligonucleotides on the flow cell, creating a bridge from one side of the sequence P5 oligo on flow cell to the other P7 , which is then amplified.
The added fluorescently-labeled nucleotides are detected using direct imaging. The sensitivity of DNA ligase to base-pairing mismatches is utilized instead, with the fluorescence produced used to determine the target sequence. Digital images taken after each reaction are then used for analysis. DNA nanoball sequencing is a form of sequencing by ligation that exploits rolling circle replication. Concatenated DNA copies are compacted into DNA nanoballs and bound to sequencing slides in a dense grid of spots ready for ligation-based sequencing reactions.
However, there are also disadvantages, including poor interpretation of homopolymers and incorporation of incorrect dNTPs by polymerases, resulting in sequencing errors. The short read lengths also create the need for deeper sequencing coverage to enable accurate contig and final genome assembly. The introduction of 3G sequencing circumvents the need for PCR, sequencing single molecules without prior amplification steps.
Depending on the method and the instrument used, some of the advantages of 3G NGS include: Real-time monitoring of nucleotide incorporation Non-biased sequencing and Longer read lengths Nevertheless, high costs, high error rates, large quantities of sequencing data and low read depth can be problematic. Similar to 3G, nanopore technology requires no amplification and uses the concept of single molecule sequencing but with the integration of tiny biopores of nanoscale diameter nanopores through which the single molecule passes and is identified.
The 4G systems currently offer the fastest whole genome sequence scan but are still quite expensive, error prone compared to 2G techniques and relatively new. Consequently, there is currently less extensive data available for the technique. Extracted samples are quality control QC checked, using standard methods spectrophotometric, fluorometric or gel electrophoretic. If using RNA, this must be reverse transcribed into cDNA, however some library preparation kits may include this step.
The optimal fragment length depends on the platform being used. It may be necessary to run a small amount of fragmented sample on an electrophoresis gel when optimizing this process. These fragments are then end-repaired and ligated to smaller generic DNA fragments called adapters. Adapters have defined lengths with known oligomer sequences to be compatible with the applied sequencing platform and identifiable where multiplex sequencing is performed.
Multiplex sequencing , using individual adapter sequences per sample, enables large numbers of libraries to be pooled and sequenced simultaneously in a single run. This pool of DNA fragments with adapters attached are known as a sequencing library.
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Next Generation Sequencing Techniques and their applications
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The reads go through quality control and are aligned to a reference genome before being evaluated for significant characteristics. The method chosen for a given experiment depends largely on what you hope to learn from the experiment. NGS Applications Whole Genome Sequencing is used when comparing genotypes and a comprehensive evaluation of the genome is needed, such as when researching rare diseases. Whole exome sequencing is a fast and cost-effective approach to interrogating protein-coding genes in a genome and is often used for tumor normal sequencing.
Each type of NGS method comes with its own unique benefits and challenges. Talk to a scientific application specialist to choose the right one for you and your experiment. This extensive application guide provides chapters on NGS workflow, as well as types of sequencing and applications.
Data analysis and new sequencing platforms are also discussed. The guide is written by IDT scientists, and is free—simply download it here. Figure 2: Diagram representing the principle 2G sequencing platforms and chemistries. Proton detection sequencing relies on counting hydrogen ions released during the polymerization of DNA. Unlike other techniques, it does not use fluorescence and does not use modified nucleotides or optics. Instead, pH changes are detected by semiconductor sensor chips and converted to digital information.
During the synthesis reactions, the fragments bind to oligonucleotides on the flow cell, creating a bridge from one side of the sequence P5 oligo on flow cell to the other P7 , which is then amplified. The added fluorescently-labeled nucleotides are detected using direct imaging. The sensitivity of DNA ligase to base-pairing mismatches is utilized instead, with the fluorescence produced used to determine the target sequence.
Digital images taken after each reaction are then used for analysis. DNA nanoball sequencing is a form of sequencing by ligation that exploits rolling circle replication. Concatenated DNA copies are compacted into DNA nanoballs and bound to sequencing slides in a dense grid of spots ready for ligation-based sequencing reactions.
However, there are also disadvantages, including poor interpretation of homopolymers and incorporation of incorrect dNTPs by polymerases, resulting in sequencing errors. The short read lengths also create the need for deeper sequencing coverage to enable accurate contig and final genome assembly. The introduction of 3G sequencing circumvents the need for PCR, sequencing single molecules without prior amplification steps.
Depending on the method and the instrument used, some of the advantages of 3G NGS include: Real-time monitoring of nucleotide incorporation Non-biased sequencing and Longer read lengths Nevertheless, high costs, high error rates, large quantities of sequencing data and low read depth can be problematic. Similar to 3G, nanopore technology requires no amplification and uses the concept of single molecule sequencing but with the integration of tiny biopores of nanoscale diameter nanopores through which the single molecule passes and is identified.
The 4G systems currently offer the fastest whole genome sequence scan but are still quite expensive, error prone compared to 2G techniques and relatively new. Consequently, there is currently less extensive data available for the technique. Extracted samples are quality control QC checked, using standard methods spectrophotometric, fluorometric or gel electrophoretic.
If using RNA, this must be reverse transcribed into cDNA, however some library preparation kits may include this step. The optimal fragment length depends on the platform being used. It may be necessary to run a small amount of fragmented sample on an electrophoresis gel when optimizing this process.
These fragments are then end-repaired and ligated to smaller generic DNA fragments called adapters. Adapters have defined lengths with known oligomer sequences to be compatible with the applied sequencing platform and identifiable where multiplex sequencing is performed.
