Question: Why does my Visium CytAssist Spatial Gene Expression library have an extra high molecular weight peak?
Answer: The expected QC trace for a Visium CytAssist Spatial Gene Expression probe-based library will appear as a single peak with an average size of 240 bp. Additional peaks larger than the desired 240 bp product are often caused by over-amplification during the Sample Index PCR (SI-PCR) step. Depending on the relative abundance of the additional peak(s), there are some considerations which we will outline here.
Slight over-amplification resulting in minor peaks consisting of daisy chained library fragments, demonstrated by the library trace below, are not problematic and should not negatively affect sequencing or assay performance.
Please note that the quantification of such over-amplified libraries by electrophoresis based methods (Bioanalyzer, TapeStation, LapChip, etc) will not be accurate. As best practices, we highly recommend using the KAPA Library Quantification Kit to measure the final concentration of your 10x library. KAPA detects diverse DNA fragments with similar efficiency, and is the method of choice for library measurements due to its sensitivity as well as its ability to specifically quantify sequenceable library molecules, i.e., those fragments that contain both the Illumina p5 and p7 sequences at the ends of the molecules. Instructions are outlined in the Visium CytAssist Spatial Gene Expression User Guide.
In more severe over-amplification cases, where the off-target peak is half of the size of the library, or greater, we recommend repeating the GEX Sample Index PCR (Step 5.2) using 25% of the remaining Pre-Amplification material. Before repeating library prep, we would recommend 1) Ensure the Cycle number determination qPCR amplification plot was interpreted properly 2) If batch processing samples during SI-PCR, ensure the cycle number is within ± 1 cycle of each other.
- To prevent over-amplification, it is important to properly interpret the Cycle Number Determination qPCR plot. Set the threshold at 25% of the peak RFU or delta Rn to determine at which Cq the sample curve crosses the threshold for each sample. Once the Cq value has been determined, round that value up to the nearest whole number and add 2 cycles.
- In addition to proper qPCR plot interpretation, it is important when performing SI-PCR to only batch process samples that are within ±1 cycle number of each other. It is not recommended to combine samples if the cycle number difference is greater than 1 to avoid library over-amplification. This may result in cycling samples with 1 less cycle than calculated.
- Sample 1: Cq - 8.5 > 9 + 2 = 11
- Sample 2: Cq - 10.7 > 11 + 2 = 13
- To keep both samples within the recommended ±1 cycle number, run both of these samples with 12 PCR cycles.
Products: Visium CytAssist for FFPE, Visium CytAssist for FF, Visium CytAssist for FxF