How long does it generally take to design and synthesize probes when customizing a hematologic oncology panel yourself?

For custom panels, I recommend planning for 6–8 weeks; do not believe that stable delivery can be achieved in 4 weeks. The design phase usually requires 1–2 weeks of communication regarding the BED file, reference version, and transcripts, synthesis and QC generally take 3–4 weeks, and with logistics and acceptance, it can easily exceed 6 weeks. For hematologic malignancies with 80 genes, if complex structural regions such as FLT3-ITD and KMT2A are included, design revisions will extend the timeline. Do not cut corners on standards; it is recommended to purchase hematologic malignancy reference materials such as those from Horizon and Jingliang to validate SNV/indel, ITD, and fusions; relying solely on the synthesizer's QC data is not sufficient. The contract should specify the delivery time after design draft confirmation and penalty clauses for delay.

The regions most prone to coverage gaps during design are high-GC regions, repetitive sequences, and homologous regions. In hematologic malignancies, typical examples include CEBPA, ASXL1, some exons of KMT2A, and the region near FLT3-ITD. It is recommended that the manufacturer first perform probe coverage simulation and, for low-coverage regions, increase probe density or adjust the design rather than waiting until after synthesis to fill gaps. A starting amount of 100 ng bone marrow/peripheral blood DNA is sufficient for hybridization capture, but attention should be paid to the tumor fraction of the sample, and sequencing depth should be increased when necessary. For the first version of the design, the manufacturer can be asked to perform pilot testing with standards and real samples, focusing on the depth and uniformity of these regions rather than only looking at theoretical coverage.

Replenishment and minimum order quantities are generally calculated by number of reactions, with common minimums of 48 or 96 reactions. If you only want to do a small pilot for the first order, you can negotiate a test package, but the unit price will be higher. After the design is archived, the replenishment lead time is usually 4-6 weeks, and the first synthesis will take an additional 1-2 weeks, so project scheduling should allow margin. It is recommended to specify in the contract the minimum order quantity, tiered pricing, and batch-to-batch consistency testing methods, and preferably lock in 2-3 batches for delivery in installments at one time to reduce batch-to-batch variability. After synthesis, require the manufacturer to provide standard validation data, then have your own laboratory re-verify using the same batch of standards, and only proceed to clinical projects after acceptance is passed.