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ASHG14 Dispatch: "We Have the Technology: Next-Generation Genomic Methods"

Amy Cullinan, Ph.D.
| Oct 24, 2014

Even we admit it. Next-generation sequencing (NGS) is such a ubiquitous and reliable tool for decoding human genetics, it’s sometime easy to forget that NGS techniques themselves also draw a starting line for innovative experimental methods and approaches. One of the most interesting ASHG sessions this year was Session 59: We Have the Technology: Next-Generation Genomic Methods. All of the presentations were well chosen, well delivered, and thought-provoking, and I’ve highlighted three of the best ones here.

Max van Min from Cergentis B.V. spoke on Targeted Locus Amplification (TLA), a comprehensive method of selectively amplifying genes and detecting all variation within, while preserving haplotype information. TLA is reliant on crosslinking physically proximal loci, fragmenting and re-ligation steps similar to chromatin capture, followed by inverse PCR and sequencing to create a map of coverage along the chromosome. Dr. van Min illustrated several examples of TLA in action, including variant data from BRCA1, identifying gene fusions from leukemia, and HLA locus haplotyping. This hypothesis-neutral method of targeted locus amplification was sequence independent and thus not affected by SNPs or indels, and should enable resequencing regions tens of kilobases long. A recent paper from many Dutch/Belgian research groups shows that the TLA method may hold promise for cancer genomic studies,

The next talk was on saturation genomics editing by multiplex homologous donor repair from Gregory Findlay of University of Wasington’s Shendure Lab. As most geneticists know, variants of unknown significance are common outcomes of clinical genome sequencing. To understand more about certain variant effects, emerging methods of genome editing are useful for maintaining the regulatory landscape of the genome and generating precise information. But single edits are not feasible when the goal is thousands. The method of saturation genome editing in conjunction with a functional assay aims to measure the function of every permutation at a given locus. The technique involves CRISPR/Cas9 cleavage plus multiplexed, homologous directed repair to look at the effects of random hexamer insertion on the transcriptome. Dr. Findlay presented data from two separate, well-studied genes (BRCA1 and DRB1) and were able to see the functional impact on disruption. Future work is to focus on noncoding regions, and further improvements on editing efficiency.

Keeping to the editing theme, Ryan Collins from Massachusetts General Hospital spoke about the potential for genome editing techniques to someday be used on stem cells. To understand the precision of genome editing, Dr. Collins subjected several types of human-derived embryonic and hematopoietic stem cells to two different targeted nuclease editing systems (CRISPR/Cas9 and TALEN) and then used NGS to examine clonal cells for off-target mutations and efficiency of editing. He was also looking at effects of various genomic RNA guides used in the techniques (dual vs. single) to see if any functional effects were seen on the genome editing. He concluded that off-target mutations related to genome editing technologies are relatively infrequent, thus confirming good specificity, but that a range of effects on efficiency could be seen with single vs. dual gRNAs, leading to a memorable quote “…anything that can happen, will happen at some frequency!”

This ASHG session clearly demonstrates the wide-open horizon of research enabled by next-generation sequencing and even a glimpse into future sequencing methods and capabilities. It was a great lineup of research, and a big thank you to all of the presenters, and to ASHG for chairing this session on methods innovation.

Referenced Papers (hyperlinked above):

de Vree, PJP, et al., (2014) Targeted Sequencing by Proximity Ligation for Comprehensive Variant Detection and Local Haplotyping. Nat Biotech 32 1019-1025. DOI:10.1038/nbt.2959

Findlay, GM, et al., (2014) Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair. Nature 513 120-123. DOI:10.1038/nature13695

Veres, A., et al., (2014) Low Incidence of Off-Target Mutations in Individual CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem Cell 15(1) 27-30. DOI:10.1016/j.stem.2014.04.020