Digital transcriptome subtraction

Digital Transcriptome Subtraction

Digital Transcriptome Sequencing

Digital transcriptome subtraction (DTS) is a computational method used in bioinformatics to identify novel pathogens by analyzing the transcriptome of infected host cells. This technique is particularly useful in the discovery of new viruses and other microbial agents that may not be detectable by traditional methods.

Overview

Digital transcriptome subtraction involves the sequencing of RNA from a sample, typically using high-throughput next-generation sequencing technologies. The resulting sequence data is then computationally processed to subtract known host sequences, leaving behind sequences that are potentially of microbial origin. This method leverages the vast amount of sequence data generated to identify rare or novel sequences that could indicate the presence of an unknown pathogen.

Methodology

The process of digital transcriptome subtraction can be broken down into several key steps:

RNA Sequencing

The first step involves the extraction of RNA from the sample, which is then converted into complementary DNA (cDNA) and sequenced. This generates a comprehensive dataset of all the RNA present in the sample, including both host and potential pathogen sequences.

Subtraction of Host Sequences

DTS pie chart showing sequence distribution

The next step is the subtraction of host sequences. This is achieved by aligning the sequenced reads against a reference genome of the host organism. Reads that match the host genome are removed, leaving behind non-host sequences.

Identification of Novel Sequences

The remaining sequences are then analyzed to identify potential novel pathogens. This involves comparing the sequences against known databases of microbial genomes. Sequences that do not match any known organism may represent novel pathogens.

Validation

Finally, the candidate sequences are validated using additional experimental techniques, such as polymerase chain reaction (PCR) or in situ hybridization, to confirm the presence of the pathogen in the original sample.

Applications

Digital transcriptome subtraction has been successfully used in various applications, including:

• Discovery of new viruses in humans and animals.
• Identification of microbial agents in unexplained disease outbreaks.
• Characterization of the microbiome in different environments.

Advantages and Limitations

DTS offers several advantages, such as the ability to detect novel pathogens without prior knowledge of their existence and the potential to discover rare or low-abundance organisms. However, it also has limitations, including the requirement for high-quality sequencing data and the computational resources needed for data analysis.

Related Pages

• Transcriptome
• Next-generation sequencing
• Bioinformatics
• Pathogen