Barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs)

Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here a team led by Ruhr University Bochum scientists presents a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In this protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA–RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool the researchers designed, the data are ready for downstream analysis. This protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans.

Laboratory workflow

Outline of the proposed Tag-Seq protocol with modifications to the nucleic acids. Sequence regions relevant for sequencing on Illumina machines are color-coded.