Question: Can library molecules be generated from Probe Hybridization events with genomic DNA?
Answer: Library reads are specifically designed to minimize the likelihood of them being generated from genomic DNA (gDNA) for several reasons:
- The Hybridization conditions won’t effectively denature gDNA.
- The templating strand needs to be degraded for the ligated probe to be released and captured by the Visium Slide, and the digestion enzyme employed in Visium FFPE is specific to RNA.
- Only molecules captured by the Visium Slide undergo the Extension reaction which adds the UMI, 10x Spatial Barcode, and partial Read 1 sequences.
- Only molecules subjected to Extension have the PCR handles required for Sample Index PCR. Molecules without the required PCR handles will not have the necessary elements required for sequencing and demultiplexing.
Therefore, not only was the Transcriptome Probe Panel design process highly stringent but there are also many protocol steps in place that make library molecules being generated from gDNA extremely unlikely.
Products: Visium for FFPE