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Next Generation Sequencing Workflow


Sample Preparation |  Fragmentation | Fragment Size Selection | Quality Control  |  Library Preparation | Template Enrichtment/ Sequencing


 

NGSWorkflowII
The next generation sequencing workflow can be divided in the sample preparation and sequencing (wet lab) and the data analysis (bioinformatics) workflow. This page focuses on the sequencing workflow. The figure provides a general overview about the NGS workflow. 

Nucleic Acid Extraction

Nucleic acid extraction is normally not a step included in the sequencing workflow but it is one of the most critical aspects that influence the final sequencing results. This is especially important in samples with low nucleic acid concentrations such as microbial samples or difficult sample material such as soil, FFPE or fecal samples. More information about nucleic acid extractions can be found on the corresponding website. We provide an extensive list of nucleic acid extraction solutions for different sample materials.


Sample Preparation

In general, the sample preparation (RNA or DNA) is the first step in the NGS workflow. Sample preparation can include cDNA synthesis (for RNA samples such as viruses or in transcriptome analysis), steps for target enrichment or depletion (for example depletion of ribosomal RNA), and targeted specific or random amplification of DNA (for samples with low nucleic acid concentrations such as microbial samples). Learn more about NGS sample preparation on the corresponding website.


DNA Fragmentation

DNA Fragmentation can be performed by different methods (enzymatic, physical shearing by centrifugation, nebulization, or sonication). The resulting fragments will further undergo end repair, barcoding or adapter ligation, followed by fragment size selection. Depending on the application and the used library preparation solution all these steps can be included in the library preparation. Sometimes all these individual steps are summarized in the library preparation step.
Learn more about DNA Fragmentation for NGS.


Fragment Size Selection

Depending on the used method for DNA fragmentation you will obtain a range of fragments of different sizes. There are a number of different fragment size selection and quality control solutions and kits available. Fragment size selection can be based on gels, magnetic beads, or spin columns.
Learn more about DNA fragment size selection on the following website.


Quality Control

Quality control steps can be performed between different steps of the NGS workflow, depending on the used methods and kits quality controls can be implemented after nucleic acid extraction, sample preparation, fragment size selection, library preparation or template preparation.
Find solutions and learn more about quality controls for NGS library preparation and sequencing on the corresponding websites.


Library Preparation

For the next step, the NGS library preparation, great variations exist between the different library preparation kits available. Depending on the used protocols, kits and individual technologies the library preparation can include various steps or only the ligation of the adaptors and/ or barcode sequences. Some library kits, however, provide a full workflow from cDNA synthesis to amplification or target enrichment and depletion, fragmentation, and adaptor ligation.
Learn more about NGS library preparation and find the best solution for your sequencing projects.


Template Preparation

Depending on the used sequencing technology, additional template amplification steps such as bead-based emulsion PCR are required after library preparation, prior to the final wet-lab step which is the sequencing itself.


Sequencing

The sequencing process is the last step in the wet lab NGS workflow and differs depending on the used technology. You can learn more about the different NGS technologies on the corresponding pages.


Data Analysis

The final step during NGS is the data analysis. This is not part of the wet lab NGS workflow and we consider this as a separate working step. More info are provided under Bioinformatics.