The Basics and Applications of Genome Sequencing

Overview of Genome Sequencing

Genome sequencing refers to the use of high-throughput sequencing platforms to sequence the genomes of different individuals or groups and perform bioinformatics analysis at the individual or group level. Genome sequencing can comprehensively mine genetic variations at the DNA level, including larger structural variations, providing crucial information for screening pathogenic and susceptibility genes for diseases, researching pathogenesis and genetic mechanisms, and inferring population migration and evolution.

Categories of Genome Sequencing

De novo Sequencing

De novo sequencing can sequence the genome of a species without any reference genome information. Through bioinformatics analysis methods for splicing and assembling, the complete genome sequence map of the species can be obtained, thereby advancing in-depth research on the species. Only through de novo sequencing can detailed genetic analysis be conducted for any organism. This method is often used for genetic analysis of species that frequently mutate, such as highly variable viruses.

Resequencing

Resequencing involves genome sequencing of different individuals of a species with an existing reference genome, followed by differential analysis at the individual or group level. After a genome has been published, resequencing does not require reassembly and can directly align the sequenced reads against the short sequences. In short, resequencing is genome sequencing of individuals of a species with a known genome sequence, primarily aimed at identifying types of variations such as single nucleotide polymorphisms (SNPs), copy number variations (CNVs), insertions/deletions (InDels), etc.

Genome Sequencing Experimental Workflow

Library Construction Process

1.Sample sources can include fresh tissues and blood, from which DNA is extracted after processing;

2. Then, genomic fragmentation is performed using enzymatic or physical methods to recover the target DNA fragments;

3. Next, end repair and A-tailing of the target DNA fragments are performed, followed by adding sequencing adapters to the adenylated DNA fragments from the previous step. Fragment selection is then done to effectively recover DNA fragments with sequencing adapters added at both ends. This is followed by PCR amplification enrichment of the captured library and then quality control of the library;

4. Finally, the library is diluted, mixed, and loaded onto the sequencing machine.

Bioinformatics Analysis Workflow

1.Data Generation

Statistics of total base count, number of reads mapped to the whole genome, and number of uniquely mapped reads, followed by sequencing depth analysis;

2. Consensus Sequence Assembly

Align the sequencing data against the reference genome sequence, using a Bayesian statistical model to detect the most likely genotype for each base site, resulting in the consensus sequence of the individual's genome;

3. SNP Detection and Distribution

Extract all polymorphic sites in the entire genome, filter and screen based on quality score, sequencing depth, and reproducibility to obtain a high-confidence SNP dataset, and annotate the detected variants based on the reference genome sequence;

4. InDel Detection and Distribution

Allow gap alignments to detect credible short InDels (insertions/deletions) during mapping, with gap lengths ranging from 1 to 5 bases;

5. Structural Variation (SV) Detection and Distribution

Detect and annotate structural variation types at the whole-genome level, including insertions, deletions, duplications, inversions, and translocations. Identify and annotate these structural variations based on the alignment results of the sequenced individual’s sequence to the reference genome sequence.

Applications of Genome Sequencing

In biomedical research, genome sequencing is widely used in genome-wide association studies (GWAS) to identify single nucleotide polymorphism (SNP) sites associated with specific diseases.

Genome sequencing also has significant applications in medicine. In 2009, Illumina launched a genome analysis suite for clinical medicine, assisting doctors in diagnosing when the cause of disease is unknown or traditional therapies are ineffective. As the cost of genome sequencing has dropped significantly in recent years, its application potential has increased substantially. In 2011, Brigham and Women's Hospital and Harvard Medical School jointly established the Genomes2People (G2P) program, aiming to integrate genome sequencing into clinical medicine.