Basics of NGS Workflow

Different platforms support massively parallel sequencing, which vary in throughput but operate based on the same underlying chemistry of sequencing by synthesis. Briefly, the workflow starts with NGS library preparation, where specific DNA sequences or adapters are ligated to genomic DNA fragments. Next, the generated library is loaded onto a flow cell for bridge amplification, where the library is copied onto the flow cell, a process commonly referred to as clustering. DNA fragments amplified onto the flow cell are sequenced through Sanger like sequencing reactions, where millions of clusters are sequenced in a massively parallel process. Lastly, millions of reads are analyzed for nucleotide identification or base calling assessment and alignment to reference sequences.

GenScript’s NGS Solutions

GenScript has developed multiple reagents to simplify the NGS workflow and optimize NGS targeted applications.

• Library preparation kits

  GenScript offers two types of library preparation kits, GenTrack2.0 and GenNature2.0, which differ in their specific chemistry. In GenTrack2.0, initial sample preparation or DNA fragmentation, by physical or enzymatic means, is followed by addition of sequencer recognizable components through ligation. In GenNature2.0, sequencer recognizable components are added via transposase enzymes in a tagmentation based method.

  

  GenTrack2.0 offers expanded capabilities including the enzyme based fragmentation module (2.1) and the repairing module (2.2). GenTrack2.0 is more suitable for applications involving shorter DNA products (e.g., PCR products), while GenTrack2.2 may be more appropriate for use with degraded (FFPE) DNA samples. Additionally, GenTrack2.3 includes a UMI module. Overall, the choice between different NGS library preparation kits will depend on the application, sample type, and automation requirements.

  Economic and Easy DNA Library Preparation Solutions

  Drop your sequencing cost | High out-put quality | Various input sample types

  
  View kits
• NGS Adapters

  Adapters play a critical role in the success of NGS library preparation and therefore the success of NGS. Poorly designed NGS adapters may affect ligation success and may be a source of biases and failures due to misassignment of reads.

  

  GenScript ensures the quality of on-shelf as well as of custom adapters through different analysis including capillary gel electrophoresis and mass spectrometry, to ensure purity above 99% and integrity, respectively.

  

  NGS Adapters

  A precise match for your molecular diagnostic application

  
  Learn more
• Capture probes library

  Targeted NGS consists on the amplification and sequencing of a specific portion of a library and may be a preferred approach to ease operation, simplify analysis, and reduce cost. Two types of targeted sequencing methods include amplicon sequencing and hybridization capture.

  • GenFisher- GenScript’s hybridization capture relies on GenFisher probes to select the target of choice for amplification and sequencing.
  

  The workflow for hybridization capture leverages biotinylated GenFisher capture probes to enrich DNA fragments of interest. For example, following targeting of Fragment-1 with biotinylated capture probes, as indicated in blue above, this fragment is enriched and purified from the rest of the library by using streptavidin beads. Once purified, the pool of Fragment-1 is amplified and sequenced.

  GenFisher custom hybrid capture panel, synthesized based on GenScript’s core semiconductor DNA synthesis technology, provide several advantages:

  • Precisely controlled probe synthesis lead to high on-target rate and uniformity
  • Compatible with column synthesized probes for fine-tuning
  • Cost-effective and consistent performance

  GenScript Target Enrichment Solutions

  Compatible for both capture and amplicon platforms | Fulfill any standard service and products

  
  Learn more
• Universal blockers

  For hybridization capture, universal blockers reduce the frequency of permissive interactions between library adapters with target probes or with other adapters, which would lead to off-target capture.

  

  GenFisher universal blockers are designed with proprietary modifications, which enable compatibility with any adapter design as well as index length.