INTRODUCTION TO NGS
Classic Sanger DNA sequencing involves determining one small piece of an organism’s sequence at a time. Next generation sequencing (NGS) uses parallel sequencing to determine the complete order of nucleotides, or bases, that make up an organism’s DNA.
NGS starts with genetic material. The sample can be either DNA or RNA that is extracted from cells or tissue. For RNA sequencing, RNA must first be converted to cDNA.
An NGS library of your DNA (or cDNA) sample is prepared. The DNA sample is fragmented into 100–300 base pair lengths called reads. In a process called sample indexing, adapters are added to the DNA, allowing the reads be sequenced and identified. The reads are sequenced and pieced back together like a jigsaw puzzle to form the genomic sequence.
NGS can be used to sequence DNA or RNA from any organism; human, animal, plant or a microorganism. NGS provides fast and accurate data to answer almost any genomics question.
Alternatives to whole genome sequencing are targeted sequencing and amplicon 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 or amplicon sequencing is called enrichment. Enrichment typically utilizes PCR steps to amplify the targeted regions of interest and is incorporated into the library prep process.
Once the NGS library is completed, included all required enrichment, it is subjected to quality control verification, and then sequenced using an Illumina NGS platform. Focused projects are sequences in-house using a MiSeq instrument at our BL-2 laboratory in Jerusalem. Whole Genome and other very large projects are sequenced using the highly cost-effective Nova-Seq instrument at the facility of our partner Novogene Singapore.
Following sequencing, the data is uploaded to an Illumina Cloud Server where it is accessed by our in-house bioinformatics specialists and analyzed. Our team will be in touch with you to provide a final report tailored to address your research questions.
LIBRARY PREP OVERVIEW
Before a DNA or RNA sample can be sequenced by next generation sequencing, the sample is randomly sheared into smaller fragments by mechanical or enzymatic methods, and sequencing adapters are added at the ends of the nucleic acid fragments. Barcodes are then added. These are short stretches of nucleotides that are used to label individual samples so multiple samples can be pooled together for sequencing.
We use premier library prep kits that best preserve sample integrity and convert nucleic acids into a sequencing-ready library with minimal loss and bias. These include a range of exceptional library prep kits each tailored to work best with a different type of sample and research application goal.
Depending upon the application, the library is then subjected to an enrichment process to facilitate targeted sequencing.
Highly focused Amplicon Sequencing for generating NGS libraries from gDNA for one or two targets, utilizes a first PCR process to generate the amplicon that will be the focus of the sequencing and a second PCR process to attach barcodes required for the NGS.
Amplicon Sequencing for up to 500 targets utilizes target enrichment by multiplex PCR using either a pre-designed amplicon panel or a custom amplicon panel. Pre-designed amplicon panels are available for genetic diseases, oncology, SARS-Cov-2, the full 16S genetic region and more.
For targeted sequencing of human or disease model samples with more than 500 targets, we utilize IDT’s Hybrid Capture panels. These panels capture the regions of interest within the library using biotinylated oligonucleotide probes. Biotinylated probes are designed to hybridize to regions of interest, such as exome, known genes related to cancer, or genes in a metabolic pathway. After hybridization of baits to the fragmented DNA or RNA, streptavidin is used to separate the probe and targeted fragment complex from other fragments that are not bound to probes.
IDT xGen Exome Research Panel v2 is a Hybrid Capture panel using 415,115 pooled individually synthesized probes and provides the most consistent performance and complete coverage of any exome panel on the market.
For custom targeted genotype sequencing of plant or animal samples with more than 500 targets, we utilize the highly efficient Allegro system from Tecan Genomics.