WYMM Tour: Cambridge, MA

Analysis of human mitochondrial tRNA using the Oxford Nanopore RNA004 platform

Miten Jain, Robin Abu-Shumays, Hugh E. Olsen, Mark Akeson

Use of Oxford Nanopore direct RNA sequencing for full-length tRNA sequencing is well documented (Thomas et al. 2021; Lucas et al. 2023; Suzuki, London Calling 2023; White et al. 2024; Shaw et al. 2024). Recently, Oxford Nanopore Technologies released an updated Nanopore direct RNA sequencing platform (RNA004) with a substantially higher accuracy (98.3% for poly(A) RNA in our hands), and higher throughput. We applied RNA004 sequencing to human mitochondrial RNA from lymphoblastoid and embryonic stem cell lines.

tRNA reads were acquired as either part of three RNA classes (tRNA + rRNA + mRNA) or using exclusively tRNA-specific adapters. In the latter case, we acquired 13 million+ tRNA reads, including 1 million+ mitochondrial tRNA reads, using PromethION flow cells. The median basecall identity was ~92%. The accuracy is lower than for poly(A) RNA due to the abundant modifications in tRNA. By comparison, median basecall identity for synthetic canonical mt-tRNA (IVT-derived) was higher for over 1 million+ aligned reads. Using these data, we were able to document alignments to all 22 known human mitochondrial tRNA isoacceptors. Partially processed, polycistronic mitochondrial RNA strands were also observed. We will discuss how these results pertain to systematic sorting of mt-tRNA isotypes, principled base modification analysis, and their utility for detection of aberrant tRNA in disease.

Next generation genome and exome sequencing technologies have revolutionized approaches to molecular diagnosis of patients with rare monogenic diseases. Depending on inclusion criteria and clinical indication, diagnostic rates typically vary between 10-50% of patients subjected to clinical or research-based testing. Traditional short-read technologies have relatively low error rates, the ability to generate high read-depth datasets, and are highly sensitive for identifying single nucleotide variants (SNVs) and small insertions and deletions (indels). However, the sensitivity for detecting structural variants (SVs), copy number variants (CNVs), short tandem repeat (STR) expansions, and other aspects of genome structure such as variant phasing, epigenetic context, and transposable elements is relatively limited. To evaluate the utility of LR genome sequencing in patients with prior short-read negative exome or genome sequencing, we conducted Nanopore sequencing on a combined cohort of incompletely or undiagnosed rare disease patients with prior clinical or research short-read sequencing studies.

The BEACON Project aims to apply Oxford Nanopore sequencing to 1,000 participants, consisting largely of parent-child trios in whom the proband was previously evaluated by short-read genome or exome sequencing. Probands were chosen from among three rare genetic disease cohorts of the Children’s Rare Disease Collaborative (CRDC) Program at Boston Children’s Hospital and individual cases were evaluated in light of available detailed phenotypic information and pre-existing short-read genomic data.

Initial data for more than 200 families have been analyzed to date. The majority of cases represent sporadic or likely recessive cases and were sequenced as trios consisting of an affected proband and two unaffected parents. The study has identified 35 diagnostically meaningful findings. This presentation will survey those results, provide a variety of examples of different findings of diagnostic relevance and discuss the evolving role of long-read genome sequencing in a molecular diagnostic context.

We developed iSCORED, a one-step random genomic DNA reconstruction method that enables efficient, unbiased quantification of genome-wide CNVs. The iSCORED pipeline represents the first method capable of high-resolution CNV detection within the intraoperative timeframe. By combining CNV detection and methylation classification, iSCORED provides a rapid and comprehensive molecular diagnostic tool that can inform rapid clinical decision. The integrated approach not only enhances the accuracy of tumor diagnosis but also optimizes surgical planning and identifies potential molecular therapies, all within the critical intraoperative timeframe.

Continuous development of sequencing technologies lead to a recent change in paradigm in clinical genetic testing, allowing comprehensive WGS to be used in first line diagnostic tests. Currently the main WGS approach relies on short read sequencing due to the highly accurate results and consistently reducing cost.

Despite its multiple advantages, there are still some intrinsic shortcomings of the short reads sequencing, including analysis of nonunique genomic regions and tandem repeats. It has been demonstrated that some genetic regions are best addressed with long read sequencing with ability to fully encompass problematic areas hence resolving both the sequence of the target regions and the length variability of the repeats.

A multitude of health conditions, predominantly neurological disorders, have been shown to be associated with pathogenic short tandem repeat (STR) expansions. STRs represent highly heterogeneous types of variants where not only repeat unit count but also composition of the repeat loci might greatly vary between individuals both in the healthy and affected population.

Compositions of the expanded repeat areas might influence the age of onset, penetrance, and severity of symptoms of some conditions, with presence of interrupting sequences acting as stabilizing factor that affects both the extent of somatic heterogeneity in the individual and repeat length expansion/contraction in transfer between generations.

Here we demonstrate that addition of long reads sequencing for elucidation of STR expansions detected with short reads, significantly improves quality of neurological disorders genetic testing results, allowing differentiation between pathogenic, intermediate and benign repeat expansions at any length range as well as determination of the repeat compositions.

At CATALOG we encode digital data into DNA, allowing for forever storage due to the stability of DNA molecules. Data that has been encoded can undergo various molecular biology processes, such as amplification, with the final readout coming from DNA sequencing. DNA molecules that encode data can reach hundreds of base pairs in length making long read sequencing a must. Oxford Nanopore Technlogies’ sequencers are essential to our processes and ensure that the entire molecule is sequenced, something that is not possible using paired-end short read sequencing.

Our current endeavor is centered around creating a fully integrated, end to end workflow for leukemia diagnosis, utilizing the Nanopore platform. This approach aims to reduce the diagnostic turnaround time from the traditional 8-10 days to 2-3 days, providing quicker insights into patient conditions and enabling more timely treatment decisions.