2.1 Overview

We usually correct errors from UMIs after RNA sequencing. What if there've been issues with UMIs before they are put for sequencing? We have put forward this significant concern and carried out our latest research endeavor on addressing bead synthesis errors through enhancements in bead design. This work can be read from the BioRxiv paper.

Question

  1. What causes this?

    Errors, including substitutions, insertions, and deletions.

  2. How can we mitigate this concern?

    Analysis and validation on reads with computationally simutated substitutions, insertions, and deletions during the generation of sequencing libraries.

Our quest for what causes this and how we can mitigate this has made us to come up with a technical plan, called anchor-enhanced technology, and validate its effectiveness through simulations.

Solution

Applying simulated substitutions, insertions, and deletions to read 1 sequences and using the reads for validation of whether or not anchor-placed reads are identified better than those that are normal.

For example, our recent study demonstrates that bespoke in silico simulations in the Drop-seq context can determine optimal positions for anchor sequences in oligonucleotide synthesis to mitigate errors. Additionally, these bespoke in silico simulations are supportive of quantifying the contribution of bead synthesis errors to the overall sequencing error profile. Based on the fact that one out of ten Drop-seq beads suffering from deletion errors according to Zhang et al., our simulations initiated with 50 molecules at one gene-by-cell type estimate around 8-14% of final sequencing reads suffering from bead synthesis-induced errors, and notably, this figure can even become doubled if the PCR error rate is elevated by one order of magnitude (i.e., 1e-4 to 1e-3).