Frequently Asked Questions
1) Can TLA determine the number of integration events per sample?
Yes, TLA will be able to determine the genomic positions of insertion sites. However, to answer this question in more detail, it is important to distinguish between clonal and heterogeneous samples.
In clonal samples, the identification of integration sites will be done based on:
- The high sequence coverage at the genomic vector integration sites, as visualized in the genome-wide sequence coverage plots (i.e., in the position of the genome where the integrations have occurred)
- The detection of the genome-vector breakpoint sequences that indicate the integration site on the single nucleotide level.
In virally-transduced samples such as CAR-T cells, with thousands of unique integration sites, TLA evaluates integration sites through the identification of breakpoints between the integrated vector and host genome. When it comes to random integrations, it is important to note that we will only be able to detect those random integration sites if they constitute > 1% of the entire population of cells. Furthermore, we can analyze < 1% if the specific breakpoints (e.g., with ITRs/UTRs) are known.
2) How many integrations sites can TLA detect?
Theoretically, there is no cap on this. More concretely, we have demonstrated in multiple studies the ability of TLA to identify thousands of integration sites. An illustration of such work is one of the examples showcased in our landmark Nature Biotechnology publication. In one of the presented case studies, TLA was used to sequence the entire HIV genome in a patient’s blood sample. Here, TLA was able to identify between >1400 different breakpoint sequences originating from different positions in the genome. These genomic lesions (i.e., breakpoints) are key genetic "signatures” that allow us to then infer the number of integration sites.
3) Would TLA be able to provide the position of integration sites and, if in coding regions, which genes?
Yes, TLA generates very high sequencing coverage across the transgene sequence and across all its integration sites. Moreover, TLA identifies integration sites at base-pair resolution and will, therefore, be able to detect the exact genomic position of the integration event.
In our TLA reports of clonal samples, we will note the presence of integration in any annotated genes. In heterogeneous samples, this information is something which can be added upon request, including information (distance and name) regarding the closest transcriptional start site (TSS).
4) Can TLA detect integration sites of both the transgene and the backbone?
Yes, TLA will be able generate high sequence coverage across the transgene sequence and will therefore not only be able to assess transgene integrity but will also be able to generate high coverage across all its integration sites to determine the positions of the integration events.
If the backbone has integrated, we will be able to see this based on the generated sequencing coverage across the entire vector sequence. Depending on the results, a follow up experiment could be performed to determine which integration sites contain the backbone sequence.
5) For targeted knock-ins, how good is the technology at detecting (rare) off-target insertions? Are there any instances where off-target were missed?
We routinely analyze samples with targeted integration, whereby off-targets are detected reliably. We always use a minimum of 2 independent TLA primer pairs that bind at different sites in the vector allowing the detection of partial integrations. In this way, TLA is more reliable in detecting and deep sequencing potential (off-targeted) integration sites.
6) Do you have experience with larger vectors such as BACs?
Yes, we would certainly be able to assist you in projects that involve the integration of large vectors such as bacterial artificial chromosomes (BACs). By adding additional TLA primer pairs, TLA can validate the integrity across the entire sequence. Some examples include the recent publication by NIH (2021), or the paper released by GSK (2020). In fact, for the analysis of large vectors (>50 kb), additional TLA primer pairs can be added to ensure sufficient sequencing coverage is generated on the vector and its integration site(s). Therefore, we are not in any way limited by the size of the integrated sequence.
7) Will the presence of ectopic plasmid DNA confound and interfere with TLA analysis? How do you discriminate variants in true genomic integration results from non-integrated ones?
Whether the presence of ectopic DNA will be an issue, will become apparent from the generated coverage profiles across the vector. This would lead to two possible outcomes:
- No or limited sequence coverage across the vector backbone sequence. This result would be ideal, and the analysis can then continue.
- High coverage (many reads) mapping to the backbone which would most likely indicate interference of the ectopic DNA in the TLA sample prep.
Should the latter occur, an additional restriction enzymatic digestion of the backbone sequence could be considered (please note that this is only possible in a limited number of cases). With that said, this depletion strategy would allow enriching relevant TLA template prior sequencing. Should this approach fail, we could alternatively try to circumvent the issue by using a site directed nuclease approach. For more detailed information, please feel free to contact us and we would be happy to discuss further about such possibilities for your specific project.
8) Do you provide the raw data from the samples you run, such that we could do further analysis in-house?
Yes, we are happy to share the raw data (FASTQ files) and/or the BAM files against a small fee.
9) Do you have offices outside of the Netherlands?
Though we are only based in the Netherlands, we support customers globally. In fact, we have almost a decade of experience helping coordinate sample shipment. Therefore, it would not be a problem for us to receive your samples from abroad and/or send you our kits.
To make the shipment process as comfortable as possible for our customers, we have recently introduced a sample pick-up service. Here, you and/or your colleagues would essentially only need to make sure that your samples are packaged and ready to be picked up, after which we will take care of the rest! This means that we will make sure that someone will come and pick up your samples at your doorstep and that your sample(s) will get safely delivered to our facility. Additionally, we will (with this add-on service) also help you arrange all the shipping-related paperwork, supply a box with dry ice during pick up, replenishing ice, include a temperature logger, etc. Please feel free to reach out to us and we would be happy to give you more information on this option.
10) What is included in your TLA kit? And what are the advantages of opting for a TLA kit?
Our TLA kit is currently priced at €9000 euros and contains all the necessary reagents for TLA preparation (excluding reagents for NGS library prep) of 24 samples. You would need to have access to a sequencer as well as bioinformatic expertise to further analyze the generated data. With that said, wetlab training is strongly recommended when purchasing a first kit.
Our kits will help lower the cost-per-sample down to €375 euros. Moreover, implementing TLA in-house will allow you and your team to significantly decrease the turnaround time to about 1-2 weeks only vs. 4 weeks (for our expedited service) and 8 weeks (for our standard turnaround time).