Why Your Hybridoma Should be Sequenced with NGS Instead of PCR

Absolute Antibody’s high-throughput Next Generation Sequencing (NGS) allows us to deliver all variable heavy (VH) and variable light (VL) domains of the hybridoma cell lines, regardless of species or isotype. Additionally, we provide a full report characterizing the hybridoma’s sequences and heterogenicity or other antibody isotypes that may be present. This gives you the ability to select the correct variable domains before moving forward with an antibody candidate.

In comparison, PCR and Sanger sequencing methods use primers based on assumptions and best guesses. With their limitations, you run the risk of sequencing only a portion of the hybridoma. For a complete and accurate reading of all transcripts, NGS is the way to go. Our team has used NGS to successfully sequence more than 2,300 hybridomas in the last two years and more than 4,400 hybridomas in total.

The PCR Gamble

Traditional methods using PCR and Sanger sequencing assume your hybridoma is truly monoclonal. However, research has shown that for more than 30% of hybridomas this is not the case. Out of 185 hybridomas analyzed, 53 had one additional LC (28.6%), 2 had one additional HC (1.1%), and 4 had one additional of each (2.2%.) (Bradbury et al, 2018). Traditional methods amplify sequences using PCR primers designed on the assumed species and isotype of the antibody. As a result, these methods can only provide one heavy and one light chain. If those are not the correct variable domains, you would not realize the problem until the recombinant antibody did not perform as expected.

Companies using PCR and Sanger sequencing methods will advertise that they sequence a few clones for each antibody to get confirmation of the sequence. This means that after running a PCR reaction, and subcloning into sequencing vectors before transformation into bacteria, they will sequence a few colonies to confirm that they have the “right” sequence. This is to exclude any single nucleotide polymorphisms introduced by the PCR, and to also exclude any occurrence of non-clonality from additional transcripts. When this method is used, you are taking a gamble on the relative abundance of the truly active transcript vs. any aberrant transcripts. The more aberrant transcripts, the less likely you are to identify the active transcript at all. With Absolute Antibody’s NGS, the chance of missing the active transcript is vanishingly small, as we sample millions of reads with hundreds of thousands mapping to the antibody transcripts.


Let’s look at a hypothetical scenario. Each yellow line represents a colony with the truly active transcript.

Think the odds are in your favor? Sequencing the incorrect transcript can jeopardize your project, and lead to a waste of time, effort, and money. These case studies demonstrate how Absolute Antibody has helped our clients sequence challenging hybridomas and overcome obstacles to antibody production.


Why Next Generation Sequencing?

In addition to accuracy, Absolute Antibody’s novel Next Generation Sequencing approach offers several key advantages compared to traditional sequencing methods such as V-region PCR or 5’ RACE sequencing. Traditional methods are low throughput and not easy to automate. With NGS, we can sequence more than 50 hybridomas concurrently, which equals thousands of antibodies per year. Less advanced sequencing is mainly limited to mouse, rat, and rabbit IgG antibodies, and requires large amounts of cells (>500,000) and high-quality RNA. Absolute Antibody’s novel NGS sequencing works for any species or isotype, requires less cells (100,000), and can often rescue contaminated or non-viable cells. Don’t gamble with your hybridoma sequences, contact us today to get started on your NGS project.


No hybridoma? No problem! Absolute Antibody offers antibody protein and polyclonal antibody sequencing. Find out more about our sequencing services here.

To learn more about Absolute Antibody and our custom services, including antibody engineering and expression, visit our website or contact us.