Sequences and Cell-free DNA Technologies

May 7, 2025 by Nate Russart

Diving into the fidelity of cell-free DNA

Cell-free DNA manufacturing is an exciting new technology in the cell and gene therapy field. There are promises of lower levels of cell-derived impurities, faster turnaround times, and the ability to handle complex sequences better than traditional e. coli-based manufacturing processes.

While cell-free DNA technologies offer these advantages over cell-based manufacturing, there are some areas where DNA produced through a process devoid of cells does not quite meet the standard achieved through plasmid-based production. Sequence fidelity is a major aspect that we think deserves a little more discussion.

When we talk about sequence fidelity, we are talking about the rate at which incorrect nucleotides are introduced into the individual DNA strands in a sample. These incorrect nucleotides are found in very low levels and have no effect on the consensus sequence produced of the product. Errors are not unique to cell-free DNA; indeed, errors or mutations are present in plasmid DNA and are the basis for evolution in living organisms.

Though e. coli has a host of proofreading and error correcting complexes that minimize the replication error rate, mistakes are made on the order of 2-5 x 10-10 per round of replication (Lee, 2012; Drake, 1991). Meanwhile, phi29 polymerase, commonly used in cell-free DNA production has error rates in the range of 10-4 – 10-6 (Esteban, 1993).

Using technologies like Sanger sequencing or simple consensus building with next-generation sequencing (NGS) will not be able to detect these errors and the resultant sequence will match the reference. Therefore, advanced methods need to be used to identify and quantify the error rate, or fidelity.

Today’s sequencing technology is known for its high accuracy, yet there are still a substantial amount of errors that are introduced through library preparation and sequencing itself. Reported error rates from Illumina devices are in the 10-3 - 10-4 range (Stoler, 2021) and much higher than the rate found in e. coli or cell-free DNA technologies.

The first advanced detection method is an NGS approach that uses unique molecular identifiers (UMIs) to tag individual DNA strands with barcodes. The sequencing reads that are generated can then be traced back to the original DNA molecule and consensus sequences can be built. If a true error is present in the original DNA molecule it will be found in all of the reads of the family with that barcode.

Errors introduced through sequencing will only be present in some of the reads from that family and can be removed from analysis. In this way you can identify and filter out many of the sequencing induced errors, however some will remain. Additionally, this method requires much deeper sequencing coverage.

In my next post, I’ll continue with another method of detecting error rates, which relies on observing phenotypic changes. You can also learn more about cell-free DNA technologies by viewing a presentation I gave at the 2025 DNA Process Development and Manufacturing Summit.

View the video presentation

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