Which library preparation kit is more reliable for low-input FFPE samples?

For 10 ng FFPE DNA library preparation, it is not advisable to focus only on the brand. Prioritize kits optimized for FFPE and low-input samples; kits that include enzymatic fragmentation and U-shaped adapters are a plus. I have used a domestic low-input kit from a certain vendor; it has a 24-reaction small-pack trial, and the lead time through a local distributor is generally 3-5 days. However, FFPE samples vary greatly, so first use the same batch of 10 ng DNA for 2 parallel library preps, focusing on the pre-amplification cycle number, whether the main library peak is at 250-350 bp, and the qPCR yield. If the yield is still low, check whether the DNA is over-crosslinked or the fragments are too short. Before library preparation, check with Qubit and a fragment analyzer rather than simply using 10 ng as the input amount. For U-shaped adapters, confirm compatibility with the subsequent capture or sequencing platform to avoid buying the wrong one.

From a supplier comparison perspective, I would list "whether an FFPE repair/single-strand repair module is included," "whether enzymatic fragmentation is included," "whether the U-shaped adapter is premixed," "whether purification magnetic beads are included," and "whether a trial size of fewer than 50 reactions is offered" in a table for selection. Low-input FFPE library preparation is prone to yield loss at the end repair and ligation efficiency steps, so one should not rely only on brand reputation; it is necessary to check whether the formulation targets degradation and cytosine deamination. It is recommended to ask domestic brands for 12/24-reaction trial sizes and require them to provide a library trace from an actual FFPE sample before it counts. Regarding lead time, distributors with stock can generally deliver within one week, while imported products may take longer. During trials, it is best to also use a good DNA quality control sample as a control to rule out issues in the handling steps.

In my experience, when FFPE library preparation fails with a 10 ng starting input, at least one-third of the cause is not the kit but the upstream nucleic acid processing and quantification. FFPE DNA often has many single-strand breaks and fragmentation, and the main fragment peak may be only around 200 bp. In this case, the enzymatic fragmentation time can be shortened or fragmentation can even be skipped, going directly to end repair, which can significantly improve library yield. After library preparation, if the library concentration is low, do not blindly increase the number of cycles first, as overamplification will reduce complexity. Regarding the kit, choose a 24-reaction small package with low-input optimization and U-shaped adapters. After arrival, use library molar concentration and qPCR yield for an initial assessment, then run a small portion on the sequencer to check duplication and coverage. For lead time, ask several local distributors; domestic products in stock can ship the same day or the next day.