Base preferences in non-templated nucleotide incorporation by MMLV-derived reverse transcriptases.

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Moloney Murine Leukemia Virus (MMLV) reverse transcriptases possess terminal transferase activity, adding non-templated nucleotides to cDNA.
• This activity enables template switching for introducing barcodes/adaptors during cDNA synthesis, crucial for single-cell RNA sequencing (scRNA-seq).
• Current limitations include unknown base preferences of terminal transferase, potentially causing inefficiencies with limited mRNA.

Purpose of the Study:

• To precisely determine the base preference of MMLV reverse transcriptase's terminal transferase activity.
• To optimize conditions for efficient template switching in cDNA synthesis for scRNA-seq.

Main Methods:

• Utilized fully degenerate oligonucleotides to probe template switching events.
• Systematically analyzed nucleotide incorporation up to ten positions downstream of the template switch site.
• Performed careful optimization of reaction conditions for cDNA amplification.

Main Results:

• Identified a strong preference for guanosine (G) at the first non-templated nucleotide position.
• Observed a significantly reduced base preference at subsequent nucleotide positions.
• Developed optimized conditions enabling efficient template switching.

Conclusions:

• The characterized base preference of MMLV terminal transferase activity is critical for understanding and improving cDNA synthesis protocols.
• Optimized conditions facilitate efficient template switching, enhancing the reliability of scRNA-seq library preparation from low-input samples.
• This work provides a foundation for more robust and efficient molecular barcoding strategies in single-cell genomics.