We're still here and working hard: NGS validation update
Many apologies for the lack of updates lately, but we have been laser focused on a number of different projects that have kept us very.. VERY busy! In September, we ran our validation samples on our MiSeq FGX next generation sequencing (NGS) instrument using the Verogen Forenseq DNA signature kit. We generated a lot of really good data and were on track to get through our planned validation quite quickly and effectively. During this, however, we found two problems. 1) The data we were generating had a lower signal quality than we expected and 2) we were having to re-work failed runs much more than expected which was not sustainable and quite expensive. Thankfully, we had a great team of people working together to identify the root cause of these problems and generate solutions! The IMF team and an army of helpers from both Verogen and NicheVision worked together as a fantastic team of partners to get things back on track. During the end of September and through the month of October (when we had hoped to run our first casework samples) we were, instead troubleshooting and exploring ways to ensure the best data possible and keep the costs as low as possible. Data Quality: We have identified several factors that led to our low signal that we experienced in our validation but we have found the main culprit is that we were running too many samples each run and will need to dial back significantly to ensure our data is as good as we can possibly make it. We were initially going to run 32 samples (maximum is 36 on a micro flow cell) each run as per Verogen’s recommendations. We want to run as many samples as possible to keep our cost/sample as low as possible. The problem is the data just isn’t as good when you run that many samples on a single run. We have identified that 12 samples per run gives us much better quality of results and, because quality of data is our most important factor, we will be using that as our standard. We will continue to explore the perfect “sample input number” but to err on the side of caution, we are going to go live with this as our “standard”. This absolutely will affect our costs and we will need to increase what we charge for NGS samples when we re-evaluate these costs with this new sample number. This will not affect any samples that are in-house as we will absolutely honor pricing as posted previously. But it’s important to know that we will be increasing our pricing as our cost increases.
In the end, the quality of the data is all that matters. Rework: I want to start by saying this problem has no effect on sample integrity. This “rework” we identified did not require us to take more sample in any way. We found that we would have failed runs that we’d have to re-prepare the final flow cell that we put on our instrument. Frankly, this was a difficult problem to find a cause for, but persistence and hard work with our Verogen partners have paid off. We identified a critical step in the process that, if we adhered to the recommendations in the Verogen protocol/developmental validation, wouldn’t reach a temperature that was needed using laboratory equipment (in this case, a heat block) that we had in our laboratory. We were forced to test heating times that were longer than recommended. After consulting the Verogen team and getting feedback from their support and R&D team, we were absolutely sure that this longer heating time would not negatively affect our samples in any way, shape or form.
We have run significant amount of samples using this new process change and are confident that our re-work rate will be more in line with what we need it to be to keep our costs down and ensure we can run the amount of samples we need without sacrificing quality. We are ready to go live with samples! We feel confident that our NGS process is of the highest quality possible. We have one last problem to tackle before we can start running casework. Our validation looks good, we’ve tweaked our process and the procedure to maximize quality and we are confident in our science. Now, we need to ensure the casework samples we have in house are “concentrated” enough to give us the best chance at getting a good result for lower level DNA samples. These samples we have in-house, ready to process, have roughly between 25 and 35ul of liquid DNA extract in it. We need 5ul of sample to run it through NGS and we want to retain 10ul to ensure we do not consume the entirety of the sample (for future science breakthroughs or other labs confirming our results). Let me give an example. Let’s say we had 1ng of DNA in a sample that was 40ul (.025ng/ul). If we were to take out 5ul of that extract for our NGS process, we would only be introducing .125ng of DNA. We need to find a way to lower the volume of our extract without changing the amount of DNA within it. We were going to use a process of “drying down” these extracts using a product called DNA Stable to ensure the quality of the extract was not affected during the dry down process. Unfortunately, the company that makes DNA stable has discontinued the product recently and it is no longer available. This will affect many in the forensic DNA community that counted on this product. We have identified other ways to manage this process and are actively working on testing to ensure that this does not negatively affect our samples. We will finalize that work this week and are confident in running casework samples immediately thereafter provided we are confident in the quality of this process. We are also working with our partners to find other creative solutions to this problem (stay tuned). We will never stop trying to find the “best process” and the “best science” to ensure that we are as effective as we can to get results on these difficult samples. It’s been a rough few weeks, but we are working hard to get this finalized. Thank you for your patience!! -Danny