Next Generation Sequencing

Next Generation Sequencing (NGS) refers to a set of advanced high-throughput technologies and techniques used to determine the precise sequence of nucleotides in DNA or RNA molecules.

It has revolutionized the field of genomics, enabling researchers to decipher genetic information at an unprecedented speed and scale. In our laboratory, we harness the power of NGS to gain insight into cancer genomics and epigenetics.

While there are several NGS methodologies available, our instrument of choice is the NextSeq 2000 system by Illumina, renowned for its robust performance and reliability.

Bosch Health Campus, Photographer: Akshay Kanakan

Illumina NextSeq2000 Sequencer

At its core, the system relies on the use of flow cells, specially designed components that capture and amplify DNA fragments for subsequent sequencing. The sequence of nucleotides is determined using Illumina's Sequencing by Synthesis (SBS) method. In SBS, fluorescently labeled nucleotides are added one at a time to the growing DNA strand during sequencing. Each added nucleotide emits a distinct fluorescence signal, which is captured by the system's detector. This signal corresponds to the specific base incorporated into the sequence. By recording these signals as the DNA strand elongates, the system can accurately determine the sequence of the DNA fragment being analyzed.

For a visual representation of this process watch this video on Youtube.

In our laboratory, we employ the NextSeq 2000 for a diverse range of applications, including:

RNA Sequencing (RNA-Seq): RNA-Seq enables us to analyze the transcriptome by sequencing RNA molecules. This method provides insights into gene expression, alternative splicing, and the identification of non-coding RNAs.

Single-Cell RNA Sequencing (scRNA-Seq): This technique provides a comprehensive view of gene expression at the single-cell level, facilitating the exploration of cellular heterogeneity and unique expression profiles.

Chromatin Immunoprecipitation Sequencing (ChIP-Seq): ChIP-Seq is employed to map protein-DNA interactions, enabling the identification of transcription factor binding sites and epigenetic modifications.

HiChIP Sequencing (HiChIP-Seq): HiChIP-Seq is instrumental in the study of three-dimensional chromatin interactions, shedding light on the organization of the genome within the nucleus.

Assay for Transposase-Accessible Chromatin (ATAC-Seq): ATAC-Seq is a technique used to study the accessibility of chromatin, which is the complex of DNA and proteins in a cell's nucleus. It allows the identification of open chromatin regions, which are associated with active regulatory elements like promoters and enhancers, providing insights into gene regulation, transcription factor binding, and the epigenetic state of the genome.

Precision Run-On Sequencing (PRO-Seq): PRO-Seq is used to determine the locations of actively transcribing RNA polymerases across the genome. It is particularly valuable for studying transcriptional dynamics and enhancer activity.

Each of these applications equips us with the ability to probe various aspects of genomics, contributing to a deeper understanding of biological processes and cancer mechanisms. The NextSeq 2000 instrument, combined with these versatile NGS methods, plays a pivotal role in advancing our research endeavors.

Equipment

NextSeq 2000

from Illumina is a sequencer system which identifies the exact order of nucleotides in a high-throughput manner.

Chromium iX

from 10x Genomics is a versatile platform that uses microfluidics technology to enable droplet-based scRNA and DNA sequencing. This technology enables scientists to analyze and understand the genetic and transcriptomic profiles of individual cells within a complex biological sample. By using Chromium iX, researchers can gain insights into the heterogeneity of cell populations and uncover valuable information about gene expression and genetic variation at the single-cell level.

TapeStation

from Agilent is a laboratory instrument that provides an automated and high-throughput solution for assessing the size, concentration, and integrity of DNA, RNA, and protein samples. It is often used to verify the size distribution and quality of NGS libraries, ensuring that they meet the specifications required for sequencing on platforms like Illumina.

Qubit

from ThermoFisher is a brand of fluorescence-based instrument and assays commonly used for the quantification of nucleic acids such as DNA and RNA. This accurate quantification is crucial during library preparation for NGS to ensure proper sample input and data quality. Qubit assays are known for their high sensitivity, which means they can accurately quantify even very low concentrations of nucleic acids. They are also specific for DNA or RNA, allowing researchers to selectively quantify one type of nucleic acid without interference from the other.

For further questions, please contact:

Dr. Zeynab Najafova, Ph.D.

Group Leader

zeynab.najafova@bosch-health-campus.com

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