Evan Eichler, Long Read Sequencing of Complex Genomes
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Evan Eichler’s work has shed light on the evolution and function of the human genome. A professor of genome sciences at the University of Washington since 2003, his lab has had a string of seminal discoveries in genomics. The first to identify a large number of segmental duplications in the human genome. Then showing these duplications are often associated with genes that are involved in neurodevelopmental disorders. Also finding that they can be caused by transposable elements (i.e. jumping DNA).
His lab’s research focuses on the evolution, pathology, and mechanisms of recent gene duplication and DNA transposition within the human genome. Particularly the functional and structural impacts of segmental duplications that give rise to new genes and recurrent rearrangements associated with neurodevelopmental delay. With the long-term goal to understand the evolution and mechanisms of recent gene duplication and its relationship to genomic instability, human disease & adaptation.
Genomic duplication followed by adaptive mutation is considered one of the primary forces for evolution of new function. Duplicated sequences are also dynamic regions of rapid structural change during the course of chromosome evolution. Long-read sequencing is a core technology to study this. Generating long continuous sequences (ranging from 10 kb to >1 Mb) directly from native DNA. Driven by instruments developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) along with companies like Element, Ultima, and Illumina entering:
For single-molecule, real-time (SMRT) sequencing developed by PacBio, DNA is fragmented and ligated to hairpin adapters to form a topologically circular molecule known as a SMRTbell. Once the SMRTbell is generated, it is bound by a DNA polymerase and loaded onto a SMRT Cell for sequencing. Each SMRT Cell can contain up to eight million zero-mode waveguides (ZMWs), which are chambers that hold picoliter volumes. A light penetrates the lower 20–30 nm of each well, reducing the detection volume of the well to only 20 zeptoliters. Fluorescently labeled dNTPs are added to begin the sequencing reaction. As the polymerase begins to synthesize the new strand of DNA, a fluorescent dNTP is briefly held in the detection volume, and a light pulse from the bottom of the well excites the fluorophore. The DNA sequence is determined by the changing fluorescence emissions that are recorded within each ZMW, with a different color corresponding to each DNA base.
Sequencing by ONT tags arbitrarily long DNA with sequencing adapters preloaded with a motor protein on one or both ends. The DNA is combined with tethering proteins & loaded onto the flow cell for sequencing. The flow cell contains thousands of protein nanopores embedded in a synthetic membrane, and the tethering proteins bring the DNA molecules toward these nanopores. Then, the sequencing adapter inserts into the opening of the nanopore, and the motor protein begins to unwind the double-stranded DNA. An electric current is applied, which, in concert with the motor protein, drives the negatively charged DNA through the pore at a rate of about 450 bases per second. As the DNA moves through the pore, it causes characteristic disruptions. Changes in current within the pore correspond to a particular k-mer (i.e. a string of DNA bases of length k) which is used to identify the DNA sequence.
Long-read sequencing has made it possible to measure gene network interactions across chromosomes in ways not previously possible. These reads capture G+C-rich regions, which are mainly found in gene regulatory regions. That yields a much more complete picture within and across species of the DNA promoter regions that regulate genes.
It also provides a way to study repetitive and complex genomic regions such as centromeric regions, long repeats and complex structural variants. Particularly short tandem repeats of a few 100 bps like Alu elements, which can run around 300 base pairs. Or LINE1 elements (up to 6 kbps).
In cancer, long-reads give a more accurate assessment of mutation patterns and genome instability. And are particularly useful for cancers with copy number aberrations and unstable genomes, such as esophageal and ovarian.
For metagenomics, long-read sequencing has had some success in host-associated microbiomes, but still needs improvements in DNA extraction of long enough molecules. Something pretty difficult for complex samples (i.e. from the ocean or soil) with many different microbial strains.
