November 30, 2021

New Approaches for Microgenomics Analyses

 

Chairmen : Alexandra Whale & Daan Noordermeer

TimeSpeakerTitle

15.00-15.05

 

15.05-15.35

Daan Noordermeer  & Alexandra Whale

Daan Noordermeer

Introduction

Enrichment of long and low-abundant DNA fragments for Nanopore sequencing and its application for the characterization of complex structural variation and 3D genome organization

15.35-16.05Alexandra WhaleSupporting microgenomic measurements in precision medicine and engineering biology using digital PCR
16.05-16.25BIO-RAD speaker: Eddy VanCollenburg*Advanced ddPCR applications and assay design strategies
16.25-16.30Break 
16.30-17.00Ronan DoyleHow reproducible are results from microbial sequencing assays?
17.00-17.30Joakim LundebergSpatial exploration of transcriptomes and genomes in tissue
Concluding remarks:  Alexandra Whale & Daan Noordermeer

 

Daan D. Noordermeer (CNRS, France):Enrichment of long and low-abundant DNA fragments for Nanopore sequencing and its application for the characterization of complex structural variation and 3D genome organization

Li-Hsin Chang, Sourav Ghosh and Daan Noordermeer

Université Paris-Saclay, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette – France

Second generation sequencing, like Illumina’s Sequencing by Synthesis, has revolutionized the characterization of genome structure/function relationships. These sequencing experiments can generate massive amounts of data, yet individual reads are mostly limited to several hundreds of base pairs. As a result, it has remained challenging to elucidate more complex aspects of genome structure, like complex genomic rearrangements or chromosome structure with single-molecule precision.

Third generation sequencing, particularly PacBio and Oxford Nanopore sequencing, has overcome this limitation by characterizing individual DNA molecules without prior amplification. Nowadays, such experiments generate thousands to millions of reads that can span tens of kilobases (kb) or more. An important challenge in these experiments has been to target this sequencing towards specific sites in the genome, thereby avoiding the need for unrealistic sequencing throughput to obtain sufficient coverage at individual loci.

Here, I will present our newly developed ELF-CLAMP protocol (Enrichment of Long DNA Fragments using Capture and Linear AMPlification). ELF-CLAMP allows the efficient amplification and enrichment of multi-kb DNA fragments with minimal knowledge of their sequence (less than 40 bp), resulting in thousands of targeted loci within a single experiment. In the ELF-CLAMP protocol, DNA fragments are first selected by in-vitro CRISPR-Cas9 cutting and site-specific T7 promoter fusion, followed by linear amplification through in-vitro transcription. The resulting long RNA molecules are subsequently characterized using direct-RNA sequencing on an Oxford Nanopore sequencer.

I will further discuss results from two applications of ELF-CLAMP: the precise characterization of repair scars at sites of structural variation in cancer cells (translocations, deletions) and the identification of 3D chromosome interaction hubs within individual chromosomes from single cells, using the combination of ELF-CLAMP with Chromosome Conformation Capture (3C).

Alexandra Whale (Measurement Institute for Chemical and Bioanalytical measurements, UK): "Supporting microgenomic measurements in precision medicine and engineering biology using digital PCR."

Digital PCR (dPCR) estimates the copy number concentration of a particular target in a sample by performing limiting dilution of the sample into discrete partitions prior to PCR so that each partition contains either no target molecules or target molecules. Following PCR the proportion of positive partitions, which initially contained a target molecule(s), are used to estimate the copy number concentration without the need of a calibration curve. This quantification approach gives a highly accurate and precise copy number estimate of a target in a sample that is SI-traceable thereby demonstrating the utility of dPCR as a reference measurement procedure. This webinar will focus on how dPCR can be used to support precision medicine using liquid biopsies and CRISPR/Cas9 gene editing in engineering biology.

Ronan Doyle (London School of Hygiene & Tropical Medicine, UK): "How reproducible are results from microbial sequencing assays?"

As the implementation costs and technical barriers fall, metagenomics and whole-genome sequencing (WGS) of pathogens from patient samples is being adopted by clinical microbiology laboratories. However, there are a myriad of bioinformatic tools and pipelines used to analyse and present the data generated. We performed two inter-laboratory studies where participants were given identical sequence data and asked to predict microbial composition and antimicrobial resistance profiles using their own bioinformatics pipelines and tools. Using this data we identify key contributors to the discrepancies in results between laboratories and make recommendations on how this can improved going forward.

Joakim Lundeberg (Royal Institute of Technology, Sweden): "Spatial exploration of transcriptomes and genomes in tissue"

The cell is a fundamental unit of life, yet we know surprisingly little about them. Specific types of cells exist in every organ, and serve specialized functions defined by the specific genes and proteins active in each cell type. Comprehensive maps of molecularly defined human cell types are underway through the Human Cell Atlas (https://www.humancellatlas.org) effort. The technologies to assemble spatiotemporal maps that will describe and define the cellular basis of health and disease are currently broadly emerging. We have developed and established the Spatial Transcriptomics technology, in which tissue imaging is merged with spatial RNA sequencing and resolved by computational means. Spatial Transcriptomics technology was the first method to provide unbiased whole transcriptome analysis with spatial information from tissue using barcoded array surfaces and has since the initial publication been used in multiple biological systems in health and disease. Recent development have shown that we also can spatially score genome integrity at scale. These and other methodological aspects will be described.

Eddy VanCollenburg is a Market Development Specialist working for the Digital Biology Group. He will be presenting Bio-Rad's droplet digital PCR (ddPCR) technology and present the use of advanced assays.

Quantitative PCR (qPCR) is one of the most widely used tools in genomics. It is so powerful and user-friendly that researchers tend to over rely on it. Every technique has its limitations, and qPCR is no exception. There are situations where a more refined PCR technique such as Droplet Digital PCR (ddPCR) could yield superior results. Eddy van Collenburg will discuss when ddPCR should be used instead of qPCR and how to ease the transition.

Email: eddy_vancollenburg@bio-rad.com

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