Question: Why do a fraction of my Visium reads contain the Template Switch Oligo (TSO) at the beginning of Read 2?
Answer: A small fraction of Visium libraries are expected to contain the template switching oligo (TSO) at the beginning of Read 2. However, if a large fraction of the library contains a portion of the TSO sequence (CCCATGTACTCTGCGTTGATACCACTGCTT) at the start of Read 2, this could indicate:
- cDNA degradation or significantly shorter cDNA than expected prior to starting the reverse transcription reaction.*
- Under-fragmentation during library construction, TSO sequence not efficiently removed from the cDNA construct during library preparation.
There can also be transcript degradation as part of the normal apoptotic pathway. This would translate to intact cells, but shortened transcripts/cDNA with the TSO hybridizing very close to the polyA tail.
*Some tissue types result in shorter cDNA despite starting with a high quality tissue section (RIN =>7) leading to an increase in the fraction of TSO reads.