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Points represent mRNA molecules that travel different distances in an electric field according to their size. Large molecules (right) are separated in Laddder-seq from small molecules (left) as along the steps in a ladder.

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

Cells can turn various genes on or off. And diseases also have characteristic patterns of gene expression. This is why scientists try to sequence mRNA molecules so as to extract further information from them. The problem with established technologies is that information was often lost along the way.

A team led by Dr. Stefan Canzar from the Gene Center Munich has now presented Ladder-seq in Nature Biotechnology: a method that leads to better results. The scientists combine changes to the sequencing protocol with computer-assisted techniques. With the additional information, Canzar’s team is able, for example, to decode the function of regulatory units of neural stem cells in the brains of mice.

Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Researchers at the Earlham Institute utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation.

The researchers identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. They show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation.

Breakdown of novel transcripts identified using ONT long reads and TALON custom transcriptome annotation

Cassette exons are previously unannotated positions. *CPAT assessment of CDS coding probability (CP ≥ 0.364). **Novel junctions are previously unannotated junctions identified between existing exonic parts. All novel assessments are relative to Gencode v29 human transcriptome annotation

This work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

Lucence, a precision oncology company using liquid biopsy to bring clarity to cancer care, is announcing the availability of an expanded version of its flagship LiquidHALLMARK liquid biopsy assay that includes both cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling. LiquidHALLMARK cfDNA and cfRNA is currently available to US oncologists as a laboratory developed test and is being utilized by physicians at NCI-designated centers across the country.

Most liquid biopsies available today interrogate cell-free DNA (cfDNA) as the sole analyte. However, for the detection of certain fusions, including those that have long intronic regions such as NTRK and NRG1, incorporation of RNA sequencing can be advantageous. Gene fusions like NTRK and NRG1 have emerged as clinically significant and actionable biomarkers that can help guide physicians to matched targeted therapies. Ryma Benayed and co-authors demonstrated that adding targeted RNA sequencing to DNA sequencing to tissue testing could obtain an additional 14% yield of clinically actionable alterations.

An internal validation study conducted by Lucence also supports this approach. Out of 112 non-small cell lung cancer (NSCLC) samples, 29 fusions were detected with a combined cfDNA and cfRNA approach in plasma, compared with 20 fusions from cfDNA alone.

“Including cfRNA in a liquid biopsy assay has a significant impact on the detection of clinically relevant fusions. This solves a major known limitation of cfDNA-only assays, which have more limited fusion detection capabilities. For example, NTRK fusions which have corresponding approved TRK inhibitors, and NRG fusions which have multiple emerging therapies, are best detected by a combination of both DNA and RNA testing. This is an exciting opportunity to help more cancer patients through broader and more accurate targeting,” said Professor Gilberto Lopes, Chief of Medical Oncology at Sylvester Cancer Center at the University of Miami.

“Lucence’s experience using AmpliMark for SARS-CoV-2 as part of the fight against the COVID-19 pandemic gave us the experience with RNA to be able to expand our assay to detect fusions in cancer. It’s a great example of how R&D developed to fight COVID can now be used for ultrasensitive cancer diagnostics,” said Dr. Min-Han Tan, MBBS, PhD, Founding CEO and Medical Director of Lucence.

Lucence’s LiquidHALLMARK cfDNA and cfRNA panel combines cfRNA profiling of 27 actionable and emerging fusions with cfDNA profiling of mutations in 80 genes, fusions in 10 genes, and somatic variants in 15 cancer types. LiquidHALLMARK is powered by AmpliMark, the Company’s proprietary amplicon-based sequencing technology, which uses a unique molecular barcode and error-correction technology to ensure test sensitivity across multiple mutation types for single nucleotide variants and fusion genes.

Source – BusinessWire