Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Tag-Seq is a high-throughput method for SNP discovery and gene expression profiling.
• Compared to RNA-Seq, Tag-Seq simplifies data processing and detects rare mRNA species.
• Reduced library complexity in Tag-Seq can lead to PCR duplicates, distorting gene expression data.

Purpose of the Study:

• To present a novel Tag-Seq protocol addressing PCR duplicate issues.
• To combine unbiased RNA library preparation with joint PCR template and sample labeling.
• To enable accurate gene expression analysis and SNP discovery.

Main Methods:

• Input RNA fragmented by hydrolysis, followed by poly(A)-selected RNA ligation to DNA-RNA P5 adapters.
• P5 adapters contain sample-specific moderately degenerate base regions (mDBRs) for PCR duplicate detection.
• P7 adapter ligation via reverse transcription, with individual i7 barcodes added during amplification.

Main Results:

• Developed a novel Tag-Seq protocol with integrated PCR duplicate removal.
• Successfully applied the protocol to Daphnia RNA samples for gene expression analysis.
• Created a free software tool for demultiplexing and PCR duplicate removal.

Conclusions:

• The novel Tag-Seq protocol effectively minimizes PCR duplicates, improving gene expression accuracy.
• This method facilitates reliable detection of rare mRNA species and SNPs.
• The protocol and associated software offer a robust solution for high-throughput gene expression studies.