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DNA Methods in Veterinary Medicine
Published in Rebecca A. Krimins, Learning from Disease in Pets, 2020
There is an increasing interest in the genomics and genetics communities to look at long-range effects on the genome that involve structural variants (SVs) such as insertions of transposable elements, duplications, inversions or deletions of large blocks of DNA at a scale finer than can be carried out using traditional microscopy. Being able to characterize large blocks of DNA is also paramount for de novo genome assembly. One method to accomplish this is to use linked-read sequencing—a technique where a few large DNA molecules are captured in oil droplets, randomly nicked and copied using DNA polymerase and specialized oligomers so that each newly synthesized DNA fragment from a long DNA template in the droplet also includes a unique molecular identifier (UMI) tag. These tagged fragments are then sequenced using the methods described above (SBS). After sequencing, the UMI tag can be used to group all related reads together and the short individual reads can be assembled into longer scaffolds. This method is used for de novo assembly of genomes from different species (e.g., Hawaiian monk seal; Mohr et al., 2017) and scimitar oryx (Humble et al., 2019) and can also be used to identify structural variants and haplotype blocks from the parents. Haplotypes are particularly useful for certain gene regions such as the major histocompatibility complex (MHC) loci that are routinely tested to determine tissue matches for transplant surgery. Figure 8.3 shows an example of a region sequenced using linked reads and for which variants have been sorted into two haplotype blocks.
Novel Methods in Preimplantation Genetic Testing: Comprehensive Preimplantation Genetic Testing
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Olga Tsuiko, Joris Robert Vermeesch
The newly developed haplotyping-by-sequencing technologies require family members for phasing and are currently not suitable for the detection of de novo mutations that occurred in the prospective parents. To overcome these issues, generic strategies for genome-wide haplotyping are currently being pioneered. For example, the long fragment read (LFR) technology can generate the long-range phased variants, achieved through the stochastic separation of long parental DNA fragments into physically distinct pools. Each pool contains a fraction of the haploid genome, which is then whole-genome amplified, fragmented, and converted to a unique barcode short-read sequencing library. Following pooling and sequencing of the barcoded DNA libraries derived from the same sample, genetic variants can be assigned to parental haplotypes [36]. Using LFR technology, blastocyst biopsies have been analyzed and 82% of all de novo changes have been detected in human IVF embryos [37]. Another approach uses a microfluidic device capable of separating and amplifying homolog chromosomes of single-metaphase cells, making it possible to retrieve haplotypes of each individual chromosome [38]. Finally, microfluidics-based linked-read sequencing technology may also open new horizons for direct genome phasing and haplotyping. The technology relies on a droplet generation system that molecularly barcodes long genomic DNA and prepares libraries, using microfluidics. The generated libraries are compatible with standard short-read sequencing. Because each droplet contains an individual DNA molecule with unique barcoded primers, short DNA fragments containing the same barcode can then be computationally linked back to each other, reconstructing long-range haplotypes [39]. Although the current high costs of these proof-of-concept methods can prohibit them from routine clinical practice, we envision a profound impact of long- and linked-read sequencing technologies on future PGT to enable direct phasing and eliminate the need for analyzing additional family members.
Novel perspectives in fetal biomarker implementation for the noninvasive prenatal testing
Published in Critical Reviews in Clinical Laboratory Sciences, 2019
Jiping Shi, Runling Zhang, Jinming Li, Rui Zhang
Microfluidic-based linked-read sequencing technology. This technology is a direct haplotype phasing method for RHDO analysis. Long DNA molecules are split into gel reads such that the genes from the same DNA molecule fragments have the same barcode markers. An individual gel bead containing about 100 molecules is functionalized with millions of copies of the genes with the same barcode [89], and the DNA molecules are amplified and sequenced subsequently. Reads that share the same barcode at heterozygous SNP positions are designated as HapA and reads with the opposite alleles at heterozygous SNP positions are designated as HapB [116]. The abundance of DNA molecules of the two haplotypes is analyzed to directly infer the fetal haplotypes without the need of the proband [40, 116]. This technology can determine the association between pathogenic mutations and DNA markers by sequencing only the fetal parental gDNA, and it does not require DNA from other affected family members; thus, it is a noninvasive technical testing platform that is suitable for a series of single-gene disorders.
Next-generation sequencing and the impact on prenatal diagnosis
Published in Expert Review of Molecular Diagnostics, 2018
Rhiannon Mellis, Natalie Chandler, Lyn S Chitty
The need for parental haplotyping may also be eliminated in future, due to the development of microfluidics-based linked-read sequencing technology – a method to directly deduce the fetal haplotype without the need for sequencing both parents [72]. Prohibitively high costs currently preclude the use of this technology but these will undoubtedly fall in time [19]. Furthermore, we may now be approaching universal applicability of NIPD without the need for an affected proband thanks to the development of a haplotype-based protocol that obviates the need for designing mutation-specific assays [31].