Next Gen Sequencing
NGS (next-generation sequencing) is a technique for determining the sequence of DNA or RNA to investigate genetic variation linked to diseases or other biological phenomena. It can be dubbed “massively parallel sequencing” because it allows the sequencing of multiple DNA strands at the magnitude of millions at once.
Samples that can be sequenced are plasmid DNA, total RNA, and genomic DNA. The choice of the sample depends on the experiment and application. For each sample type, library preparation varies. In general, in long sequences such as gDNA or plasmid, the samples are fragmented. Adapter sequences that are complementary to the sequences etched on the flow cell are added through strand displacement PCR reaction. The libraries are cleaned up, diluted, and denatured before being loaded on Illumina’s proprietary cartridge.
Denatured library fragments (single-stranded) having adapter sequences binds to the flow cell owing to the complementarity between adapter in library fragments and etched sequences on the flow cell. Iso-thermal amplification, called bridge PCR, replicates the bound library fragments by taking 3’ end from etched sequences. This generates clusters of the same library fragments on the flow cell. After cluster generation, fluorescently labeled dNTPs are added sequentially and the fluorescence is read after the addition of each dNTP type. Clusters incorporating a particular dNTP at the Nth cycle represent that base at the Nth position in that library fragment.
The machine analyzes the images and gives output in the form of fastq files that contain library fragment sequences and per-base quality information. Raw files are assessed for quality and processed as per application. The raw sequences can be assembled de-novo or aligned to the pre-existing reference sequence.
At present, we have optimized a pipeline for de-novo assembly, variant calling, annotation, and Gene ontology clustering.
DBT supported Genomic DNA sequencing facility houses Illumina’s MiSeq system. This national facility with state-of-the-art instrumentation serves national R&D and academic establishments having DSIR authorization and Customs exemption such as colleges, public labs, government foundations, and not-for-benefits associations.
The facility has been exploited by several users across the country for their experiments.