First-time long-read sequencing of single cells

Samplix sponsored talk at ESHG 2021

August 28, 2021 at 13:45 CEST

If you are a registered ESHG attendee, you can access the session recording

Long-read whole genome analysis of human single cells

Dr. Adam Ameur from Uppsala University and Science for Life Laboratory (SciLifeLab) leveraged the unbiased droplet multiple displacement amplification (dMDA) step of Xdrop® to prepare long fragments of genomic DNA from a single cell for HiFi PacBio sequencing. This application of long-read sequencing to single cells, once believed to be impossible, revealed structural variation beyond what is visible with short-read sequencing.

Abstract:

Long-read sequencing has accelerated the completion of genome assemblies and elucidated complex genetic variation. This work demonstrates the first application of long-read technology to examine the genomic architecture of individual human cells. DNA from single CD8+ T-cells was amplified via a novel droplet-based multiple displacement amplification (dMDA) method enabled by the Samplix Xdrop technology that ensures uniform coverage of genome-wide long DNA molecules with minimal bias. Amplified DNA from two single cells was sequenced on the PacBio Sequel II system, generating over 2.5 million reads per cell and up to 40% genome coverage. Compared to data from short-read sequencing of clonally related cells, the long-read sequencing data uncovered comparable numbers of single nucleotide variants and four times as many structural variants. Furthermore, the long-read data revealed somatic structural variation between analyzed T-cell clones and enabled de novo assembly of parts of the single-cell genomes. The method presented goes far beyond limits of current long-read sequencing protocols, which normally require at least 5 ng of input DNA. In summary, coupling uniform whole-genome amplification in droplets with high fidelity long-read sequencing is a viable approach towards unravelling the extent and distribution of complex genetic variation at the single-cell level.

A Ameur

Photo credit: Uppsala University

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