title: Digital gene expression analysis with sample multiplexing and PCR duplicate detection: A straightforward protocol.
abstract: 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 we present 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 our 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 we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans.
authors.affiliation: Department of Animal Ecology, Evolution and Biodiversity, Ruhr University Bochum, Bochum, Germany.
authors.forename: Andrey
authors.initials: A
authors.lastname: Rozenberg
authors.affiliation: Faculty of Biology, Aquatic Ecosystem Research, University of Duisburg and Essen, Essen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Leese
authors.affiliation: Environmental Genomics Group, School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Linda C
authors.initials: LC
authors.lastname: Weiss
authors.affiliation: Department of Animal Ecology, Evolution and Biodiversity, Ruhr University Bochum, Bochum, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Tollrian
mesh_terms: D000818:Animals; D003621:Daphnia / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012313:RNA / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D012984:Software
chemical_list: D012313:RNA
keywords: Daphnia; PCR duplicates; RNA library preparation; SNP discovery; Tag-Seq; digital expression analysis; inducible defenses