Everett Weinstein Intern GEN
Utilization of Cas9 to Enhance Efficacy of Nanopore Sequencing
A group of scientists from Tel Aviv University recently performed an experiment utilizing Cas9-Assisted Targeting of CHromosome segments (CATCH) to isolate a gene typically associated with breast cancer, BRCA1, and used nanopore sequencing to map the gene.
The team extracted 200kb strands of DNA from primary human peripheral blood cells—containing the 80kb BRCA1 gene and its adjacent sectors—and proceeded to process the strands with long-range amplification. Once the target sequence of 200kb was sufficiently enriched, the team began nanopore sequencing which allowed them to determine the order of nucleotides that comprised the section of interest, and even allowed for the registration of both coding and non-coding areas which may help shed light on less straightforward genetic determinants.
Being able to extract, amplify, and analyze only 200kb rapidly expedites the ability to sequence a specific area within the human genome as compared to the massive 3 billion base pairs that make up all of our genetic information. “Due to high sequencing costs, cutting out only the region of interest from the genome focuses all your sequencing efforts and money toward this target without ‘wasting’ reads on DNA from the rest of the genome,” explains Yuval Ebenstein, Ph.D., a professor at Tel Aviv University.
Techniques for mapping the genome are varied, including single-molecule real-time (SMRT) sequencing, nanopore sequencing, and, a little older but still relevant attributable to its higher accuracy, next-generation sequencing (NGS)—an umbrella term for technologies such as Illumina sequencing.
However, while these methods have proven effective they are not without their limitations. NGS is hindered due to its inability to process structural aberrations greater in size than a few hundred base pairs. Long-read methods such as SMRT and nanopore sequencing are capable of detecting variations that NGS can’t, and both provide real-time viewing of data. Nanopore sequencing takes it a step further and is capable of analyzing native DNA without the need for PCR amplification techniques—which all other sequencing methods depend on. The elimination of prerequisite amplification is a huge advantage because it is often during PCR amplification that more errors and single nucleotide polymorphisms (SNPs) are produced. With that said, nanopore sequencing still has a ways to go in terms of achieving greater accuracy, while NGS has shown itself to be tried and true.
Dr. Ebenstein points out that while nanopore sequencing is normally able to make use of native DNA, “CATCH isolates only a tiny fraction of the genome which means that very low amounts of DNA are recovered for library preparation. Currently, this implies that the DNA must either be isolated from a very large amount of cells or amplified to achieve the needed amount of material for sequencing.”
Finance, Function, and Future
What does all of this mean? NGS is incapable of processing reads bigger than a few hundred base pairs, while nanopore technology is capable of processing reads up to several kilo base pairs. However, an intrinsic obstacle to nanopore sequencing is that it can be expensive. It’s true that the upfront cost for Oxford Nanopore Technologies’ MinION retails for only $1,000, but while “the initial capital investment is indeed lower, the price per base on the MinION is still considerably higher than NGS,” notes Dr. Ebenstein, “although [nanopore sequencing] can obtain information that is inaccessible to NGS.”
By utilizing CATCH, scientists can isolate and enrich smaller portions of the genome, as done in this experiment, by having the Cas9 cut at the sequence of interest’s flanking regions. It is possible to expedite the process, as nanopore sequencing can be used only on the cut-out region of interest rather than the entire genome. That was one of the crucial points that the team wanted to convey with this experiment.
“CATCH brings nanopore sequencing closer to the clinic by offering targeted, cost-effective sequencing,” explains Dr. Ebenstein. “You only need sequence knowledge for two regions flanking your target, thus enabling the enrichment of large variable regions in the genome that are very complex and challenging for conventional approaches.”
This is cause for excitement as CATCH furthers the potential of an already impressive technique for DNA sequencing. Nanopore sequencing is already highly valued for its ultra-long reads and real-time analysis capability, but with the incorporation of CATCH the entire process can occur even faster and with greater fiscal efficacy.
“The long reads provided by nanopore sequencing allow for the characterization of structural variations in conjunction with smaller aberrations such as SNPs. Together, this information is critical for understanding the genetic causes of disease,” states Dr. Ebenstein.
The results of the experiment were telling as to the capabilities and limitations of both NGS and nanopore sequencing. In their paper, published in Nucleic Acids Research as “Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)”, the team shared data that “NGS discovered 177 SNPs. Nanopore data revealed 62% of the SNPs with only two SNPs not present at all in the nanopore data and the remaining 37% inconclusive.”
“NGS has a lower error rate compared to nanopore. Therefore, it is more qualified to detect single base mutations,” says Dr. Ebenstein. “On the other hand, sufficient coverage (offered by CATCH) can overcome these errors by averaging over many reads. In addition, nanopore has better access to larger aberrations such as structural and copy number variants.”
Yael Michaeli, Ph.D., School of Chemistry, Faculty of Exact Sciences at Tel Aviv University, mentions that their team “used a software that is more compatible with NGS reads and this in part may explain the inferior results of the nanopore.”
It’s clear that there are still some areas of uncertainty surrounding nanopore sequencing; and compared to the competition it still has to prove itself a little further in terms of reliability. Still, Dr. Ebenstein believes that “by now it seems quite clear that nanopore sequencing will find its place in routine genomic analysis pipelines. [However,] it is still more expensive than NGS, and should be chosen only when needed.”