Composition, Purpose, and Goals
The "Genomic Medicine Core Laboratory" began its services on January 1, 106 (2017).
The "Genomic Medicine Core Laboratory" integrates the existing Core Laboratories for Gene Sequencing, Genomics, and Bioinformatics. Its main research focus is RNA-guided gene expression analysis and the analysis of disease-related gene variations through genomic analysis.
The mission of the "Genomic Medicine Core Laboratory" is to:
1. Provide state-of-the-art genomic research equipment to Chang Gung Memorial Hospital and Chang Gung University researchers.
2. Assist researchers in the Chang Gung system in designing genomic medicine experiments and provide data analysis consultation through bioinformatics.
3. Provide education and technical training in genomic medicine to researchers and students in the Chang Gung system.
4. Establish an open forum for researchers from various fields to discuss and collaborate.
Core Technologies
1. Illumina MiSeq System
The Illumina MiSeq sequencer is a desktop sequencer. It provides high-quality data and allows users to choose various sequencing throughputs and read lengths based on experimental needs and budget considerations. The MiSeq system is suitable for small genome sequencing (bacteria, viruses), 16S metagenomics microbial community analysis, targeted gene panel sequencing, chromatin immunoprecipitation sequencing, microRNA sequencing, and methylation analysis.
With the increasing amount of gene sequencing data and genomic databases, the importance of data organization, analysis, and studying the relevance of diseases will significantly increase in biomedical research experiment design. Bioinformatics is a new research field that combines biology, computer science, and information technology. The ultimate goal is to discover new biological insights and establish a comprehensive understanding of biological systems. Next-generation sequencing analysis integrates massive data (Big Data) from different levels in an informatics approach to help researchers gain a deeper understanding of the pathogenic factors and mechanisms of genomic variations in diseases. The analysis of next-generation sequencing data (raw FASTQ files) requires computational programs. The initial analysis involves sequence alignment and variant calling, commonly using alignment programs such as BWA, Bowtie, and Bowtie2. The resulting data is stored in BAM files, and variant calling programs such as GATK and FreeBayes are used to identify single nucleotide variants (SNPs) and small insertion/deletion variants (INDELs). The programs also calculate variant read counts, quality, and genotype determination, and the data is written to VCF files. Functional annotation of variants is performed, and standard analysis pipelines can be established for repetitive analysis with the same conditions or for process improvement. The ultimate goal is to integrate different omics data to identify gene regulation, protein interaction networks, cellular signaling pathways, metabolic networks, and drug actions, providing therapeutic strategies for disease problems at a systems level. The Core Laboratory has established a next-generation sequencing data analysis platform (CLC Genomics Workbench) environment to provide researchers in the Chang Gung system with a comprehensive research facility. The goal is to establish a complete next-generation gene sequencing and cloud database for clinical and basic biomedical research, conduct integrative systems biomedical research, and provide gene sequencing data processing and exploration.
2. ABI 3730
The ABI 3730 capillary electrophoresis DNA sequencer utilizes the Sanger dideoxy chain termination method. It involves the use of DNA as a template, along with the addition of appropriate dNTPs, DNA polymerase, and four types of fluorescently labeled nucleotide analogs (ddNTPs: ddATP, ddTTP, ddCTP, ddGTP) in a test tube for the polymerase chain reaction. Since the nucleotide analogs lack a 3-hydroxyl group, they cannot form a phosphodiester bond with the 5-phosphate group of the next nucleotide. This results in the termination of DNA synthesis at positions where the nucleotide analogs are incorporated, leading to DNA fragments of varying lengths. These DNA fragments of different lengths are then separated using high-resolution capillary electrophoresis. Finally, the DNA sequences of these fragments, which differ in size, are read through laser excitation and detected by a CCD camera. The computer then assembles the DNA sequences to decipher the complete sequence.
3. Microarray
A DNA microarray (also known as a DNA chip) is a surface-treated chip where DNA molecules are systematically immobilized. In DNA microarray analysis of gene expression, the messenger RNA (mRNA) is first converted into complementary DNA (cDNA), which then hybridizes with the DNA immobilized on the microarray. The hybridization process occurs through Watson-Crick base pairing. The "probes" are known nucleotide sequences that are orderly placed on the surface of the chip, while the "targets" are nucleic acid molecules obtained from biological samples. The primary utility of DNA microarray chips is their ability to analyze gene expression on a large scale, simultaneously examining thousands of genes.
Currently, there are two basic types of DNA microarrays, namely "oligonucleotide microarrays" and "cDNA microarrays," which differ in their manufacturing methods and the substances attached to the chips surface.
Some oligonucleotide microarrays have the oligonucleotides directly synthesized and arranged in a well-defined pattern on a silicon or glass chip. In contrast, others involve the synthesis of oligonucleotides before spotting them onto glass slides. On the other hand, cDNA microarrays involve fixing prepared DNA clones onto a glass slide. These DNA clones are amplified products generated through polymerase chain reaction (PCR), typically ranging in size from 300 to 2000 base pairs.
In the late 1980s, Stephen Fodor and his team developed oligonucleotide microarrays, known as Affymetrix chips, using photolithography. Each target RNA is probed by oligonucleotides consisting of 11 to 20 base pairs. Each probe pair is approximately 25 base pairs long and perfectly complements the target RNA sequence, referred to as the perfect match (PM). Within each probe pair, there are some variant bases, typically a variation at the 13th base, known as the mismatch (MM), which serves as a control for signal analysis. The fabrication of Affymetrix chips involves the independent automated arrangement of oligonucleotide sequences on the chip until all four nucleotides occupy each quadrant of the chip. This process is repeated until the probe lengths on each chip range from 20 to 25 base pairs. In recent years, scientists have developed alternative methods to directly synthesize oligonucleotides on the chip. For example, Agilent Technologies utilizes inkjet printer heads to directly spot nucleic acids onto glass slides (http://www.chem.agilent.com). Another emerging technology, NimbleGen Systems, employs digital light processing and rapid, high-throughput photolithography to synthesize high-density DNA microarrays with oligonucleotides reaching lengths of up to 50-70 nucleotides (http://www.nimblegen.com).
In the mid-1990s, Pat Brown and his team developed the dot blot microarray method. This type of microarray involves three steps: (1) preparation of plasmid DNA clones, (2) automated spotting of DNA onto a glass slide, and (3) post-processing of the slide (UV irradiation and drying). In cDNA microarray analysis of gene expression, the following four steps are typically performed: (1) sample preparation and fluorescent labeling, (2) hybridization reaction, (3) washing to remove impurities, and (4) scanning and analysis of the images. In a dual-color system, two sets of different messenger RNA (mRNA) samples, one labeled with green (Cy3) and the other with red (Cy5) fluorescence, are often used. One sample serves as the control group, while the other represents the experimental group. These two samples are hybridized together on a glass slide containing cDNA probes. After washing, the slide is scanned using a scanner with two-color lasers, resulting in two monochrome images. The detector measures the ratio of the two labeled hybridization signals, and the results are presented on the chip as two different-colored images, typically red and green.