Genome search: the search for the invisible

Figure 1: Ladder-seq uses mRNA length information to aid in transcriptional reconstruction. Credit: DOI: 10.1038 / s41587-021-01136-7

LMU researchers have developed a method to extract more information from the sequence data. This will provide deeper insights into biology.

Higher organisms store their genetic material in the nuclei of cells in the form of deoxyribonucleic acid (DNA). In a process called transcription, individual segments, genes, are converted into ribonucleic acids (mRNAs). Then, the translation process produces proteins as the most important functional units.

Cells can turn on or off different genes. Diseases also have distinct patterns of gene expression. This is why scientists are trying to sequence mRNA molecules to extract more information from them. The problem with established technologies is that information is often lost along the way.

A team led by Dr. Stefan Kanzar of the Munich Gene Center has now presented Ladder-seq in nature biotechnology: method that leads to better results. Scientists combine changes in the sequencing protocol with computer-assisted techniques. Using the additional information, Kanzar’s team can, for example, decode the function of the regulatory units of neural stem cells in the brains of mice.

Puzzle with additional information

“In the previous sequence now, the first step was to break down the mRNA molecules, leaving you, figuratively speaking, with mixed pieces of a jigsaw puzzle,” Kanzar explains. Then modern devices arrange these parts in parallel, which saves time. Using powerful computers, individual parts of the sequence are put back together again. Over the past ten to fifteen years, many methods have been improved. “However, we know that information is lost in the process that cannot be reconstructed,” says the LMU researcher.

Dr. Francesca Rojas Ringling and Chunak Chakraborty of the Kenzar group modified the usual protocol. Before the actual sequencing, researchers separate the RNA molecules based on their length. The easiest way to do this is with a technique called electrophoresis to separate molecules in an electric field. Depending on their size, mRNA molecules travel different distances. In gel, it appears as a ladder, hence the name Ladder-seq. Only then do researchers implement hashing and sequencing. Algorithms use information about size to piece the individual pieces of the puzzle together more precisely than was previously possible.

Sample application in neuroscience

Using the sample application, LMU researchers demonstrated what Ladder-seq can achieve. In the experiment, they looked at alternative splicing in the developing mouse brain. If you remove a chemical modification of RNA, the sequence of the RNA molecule itself changes. This means that you get alternate braiding. From the same gene, a different mRNA is produced – and thus a different protein, which lacks certain structural domains. They no longer fulfill their biological roles in the brains of mice. Neural stem cells remain at this stage of development. They no longer differentiate into neurons, because the corresponding genes are dysregulated. The brains of severely infected rodents are deformed and the creatures die soon after birth. “Without the new method, we would never have been able to answer such questions with such precision and detail,” Kanzar explains.

Find out exactly what the genes say – and where

more information:
Francisca Rojas Ringeling et al, Segmentation of RNA by length improves transcriptional reconstruction from short-read RNA-seq data, nature biotechnology (2022). DOI: 10.1038 / s41587-021-01136-7

Presented by Ludwig Maximilian University of Munich

the quote: Genome Research: Finding the Invisible (2022, Jan 11) Retrieved Jan 12, 2022 from

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