NGS workflow

Before you start

Next generation sequencing is based on genetic material. Before you can prepare libraries, you must have isolated and purified genetic material. Check your DNA or RNA extraction kit for compatibility with NGS before extraction. Extraction and purification must result in adequate yield and quality for successful sequencing. For RNA sequencing, RNA must first be converted to cDNA.

Step 1: Library prep

The library preparation step allows your samples to be processed on your sequencer. IDT products are compatible with many types of sequencing platforms, including Illumina, Pacific Biosciences, and Oxford Nanopore. The library prep step fragments the sample into shorter sequences to combat sequencing bias and attaches adapters to make the sequence compatible with the sequencer. Identifying sequences called “barcodes” or “indexes” can also be added to the samples. Barcodes allow the combining of samples, also called “pooling” or “multiplexing,” so that they can be sequenced simultaneously, saving time and money.

Optional Enrichment

An alternative to whole genome sequencing (WGS) is targeted sequencing. Instead of sequencing the entire genome of your sample, you can choose specific portions of the genome to focus your study for a faster, more efficient experiment. Preparing libraries for targeted sequencing is called enrichment. Enrichment can occur either during library prep (amplicon sequencing) or after library prep (hybridization capture), depending on the enrichment method chosen.

Step 2: Sequencing

There are many types of sequencing methods. The most popular, and the method compatible with IDT products, is provided by Illumina. Illumina’s method uses sequencing-by-synthesis. Fluorescently tagged bases emit a signal as they are added to a nucleic acid chain during synthesis. Illumina sequencing provided the highest throughput, largest data output, and lowest reagent costs. Not sequencing with Illumina? We also help customers design custom solutions for other sequencing platforms.  

Step 3: Data analysis

There are many ways to analyze sequencing data, just as there are many methods of sequencing. First, accuracy must be determined through quality control. The short sequences of nucleic acids, or reads, are assigned to the appropriate samples based on the sample index, called demultiplexing. Then the adapter sequences are trimmed from the sample sequence, and the reads must be aligned to a reference genome. You can analyze the data using bioinformatics tools or data analysis apps. Each analysis pipeline and tool has benefits and caveats that come with its use.