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UCSF Group Wins NHGRI Funds to Develop High-Throughput Single Cell Digital Sequencing Methods

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NEW YORK (GenomeWeb) – Researchers at the University of California, San Francisco have been awarded funding from the National Human Genome Research Institute to develop methods for accurate and unbiased single-cell sequencing using digital droplet multiple displacement amplification, or sc-ddMDA.

The three-year funding, which was announced earlier this month and totals $713,363 for the first year, is in the form of an R01 grant to physicist Adam Abate, whose UCSF lab focuses on droplet-based microfluidics.

Droplet-based methods are increasingly being used for amplification prior to single-cell genomic sequencing. However, existing amplification techniques can also introduce biases and errors. The UCSF group is now developing biochemical and microfluidic strategies that aim to limit these undesirable effects.

Abate co-founded Mission Bio in 2013 to commercialize PCR-activated cell sorting, as previously reported. The current project is not a formal collaboration with that company, although in an interview with GenomeWeb Abate said there is scientific overlap between the newly funded research and the previous work, such that there is a possibility for some synergistic opportunities with the firm.

The proposal contrasts amplification using multiple annealing and looping-based amplification cycles, or MALBAC, with MDA, both being methods that improve upon conventional PCR. "It's not that one is necessarily better in all cases than the other, but they are different methods and have different strengths and weaknesses," Abate said.

Ideally, sequencing requires base-pair reading accuracy as well as quantitative accuracy so that the final product reflects the true proportions of sequence motifs in the original sample, Abate said. MALBAC was originally developed to help conserve quantitative accuracy, heading off amplification biases by using primers that loop and prevent the reaction from being exponential.

But the Bst polymerase used in the MALBAC method is also somewhat error prone. On the other hand, one of the strengths of MDA is that the Phi 29 polymerase it employs is known to be highly accurate and processive, "So you get long amplicons that are typically very accurate in terms of their sequence," Abate said. The MDA method, however, is not very quantitatively accurate because there is no curtailing of the exponential amplification reaction.

"This is a well-known problem with MDA and you end up with substantial bias," Abate said, adding, "You can take a single-cell genome in which every portion is present at exactly one copy, and if you just do MDA on it you'll find that you only sequence a very small fraction of the genome."

One simple solution to the bias problem is to not let the reaction go too long, he said, and this has been accomplished by Abate and others via confining the reactions to microfluidic volumes.

"Rather than sort of living with the bias, but just stopping the reaction to limit it, the other way to go is to try to get rid of the bias entirely but still have the accuracy of Phi 29 polymerase," said Abate.

The big insight was to realize that bias is fundamentally resulting from different molecules in the sample competing with one another for amplification. "One theoretical solution to bias is to prevent competition ... by performing not one MDA reaction, where all the molecules are in a single reactor, but instead perform millions of parallel MDA reactions where each molecule is in its own little reactor," Abate said.

In this case, a slowly amplifying molecule can still catch up, and the final concentration of each molecule will essentially depend on the size of the tiny reactor.

To make a million identical reactors, researchers like Abate have used microfluidics to form millions of equally sized droplets.

In a recent Nucleic Acids Research study, Abate and his colleagues showed that their digital droplet MDA method was able to amplify the genomes of single Escherichia coli cells — each with only about 5 femtograms of starting DNA — with coverage distributions rivaling unamplified material. The researchers have also recently described this process in a Nature Communications article which details the microfluidic workflow and illustrates a microfluidic technique for barcoding droplets to aid in assembly and variant calling.

In terms of the novel biochemical methods, some of it is the subject of a possible patent, Abate said, but the overall goal is to apply the droplet MDA approach to single cells in a format that is convenient and reliable.

"To really make a broad impact ... we'd like to utilize similar types of amplification principles but not have to have such fancy microfluidics," Abate said.

Abate's lab now aims to develop both an automated, high-throughput version of its method as well as a version that uses no special equipment and is accessible to any molecular biology lab. The group is also continuing its collaboration with microbial genetics experts at the Department of Energy's Joint Genome Institute to develop microfluidics-based sample prep technology for next-generation sequencing applications.

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