Clinical OMICS

MAR-APR 2018

Healthcare magazine for research scientists, labs, pathologists, hospitals, cancer centers, physicians and biopharma companies providing news articles, expert interviews and videos about molecular diagnostics in precision medicine

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24 Clinical OMICs March/April 2018 www.clinicalomics.com Improved Reference Genomes: Tools for Alleviating Reference Bias Currently, new human genome samples are mapped relative to a single, standard- ized reference genome—the one that was originally sequenced by the Human Genome Project and is now in its 19th it- eration (GRCh38). As a result, "it's easier to find variants that are in the reference ge- nome than to find ones that aren't," said Benedict Paten, Ph.D., assistant professor of biomedical engineering at the Universi- ty of California, Santa Cruz. This "reference bias" further exacerbates the challenge of identifying structural variants: If a new sample contains a structural variant that doesn't exist in the reference genome, it won't be mapped. Various ways of addressing reference bias, are in the works. Graves-Lindsay, for example, is sequencing multiple individu- al reference genomes. To make sure these new references are of high quality, her team looks at each person's DNA through multiple lenses: PacBio long reads; Bion- ano optical sequencing; 10X Genomics' linked reads; as well as additional ap- proaches including Hi-C. Once multiple individual references have been created, however, a researcher or clinician will have to decide which ref- erence to use for a given genome sample. "In an ideal world, you could align your sample to 10 different genomes and pick the one that's the best," Graves-Lindsay said. "But I don't know if clinicians are go- ing to want to do multiple alignments, and you still might have some portions of a person's genome that map better to genome 2 than genome 1." One solution to this problem is to in- clude all known human genomic variation in a single graph. This is Paten's approach, and it is one that is being commercialized by Seven Bridges Genomics. In theory, it should lead to better discovery of varia- tion in new samples. "We've convinced ourselves that we're as precise and accu- rate as other [mapping] tools and can call additional variants not available through other tools," said Deniz Kural, Ph.D., co-founder and executive chairman of the board Seven Bridges. One exciting application area is cancer genomics. Today, geneticists say theyy can compare a patient's cancer genome to his or her normal genome, but in fact they compare each sample to the ref- erence genome and then compare the comparisons. "But with graph genomes," Kural said, "you can actually use the nor- mal genome as the reference, and directly compare the cancer genome against that as well as all of the other genomes in the graph." Furthermore, the primary tumor can serve as the reference for a metastat- ic clone to better pinpoint any changes. —Katharine Miller n (continued from page 22) changes. They accomplish this by starting with a long (up to 2.5 mil- lion base-pair), intact DNA molecule. To it, they attach a fluorescent dye to a six- or seven-base-pair sequence that recurs throughout the genome of every species. Running this molecule through a nano-channel array pro- duces a scan of the molecule and its pattern of fluorescent labels. "Because the molecules are so long, and we have these labels every 6,000 bases in the genome, we end up with what looks like bar codes," Bocklandt said. When these are aligned pairwise with one another and the barcode for the reference genome, SVs stand out. Bionano's approach is often con- sidered complementary to SRS as well as PacBio and 10X sequencing, said Graves-Lindsay. And, Bock- landt added, it's particularly good at detecting the repetitive sequences that make up two-thirds of our genome. Inversions and other variants inside repetitive sequences are particularly invisible to other approaches, he said. In a recent case published in Genome Medicine last year, researcher Eric Vilain, M.D., Ph.D., of the Children's National Health System in Washing- ton, D.C., used Bionano technology to find both single and multiple exon deletions up to 250,000 base pairs in size, a 13,000 base-pair duplication, and a 5.1 million base pair inversion disrupting the dystrophin gene of several study patients with Duchenne A human genome graph consists of all the variant information for a cohort in a single graph struc- ture. Each branch has an allele frequency associated with it, measuring how often it appears among the individuals included in the graph. A specific individual's genome can be represented as a path snaking through the graph. Seven Bridges Genomics "Because the molecules are so long, and we have these labels every 6,000 bases in the genome, we end up with what looks like bar codes." —Sven Bocklandt, Ph.D., Bionano Genomics

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