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.

MicroRNA-106b Regulates Milk Fat Metabolism via ATP Binding Cassette Subfamily A Member 1 (ABCA1) in Bovine Mammary Epithelial Cells

“…The constructed library was sequenced with an Illumina Hiseq 2500 at a sequence read length of 1 × 36 bp. 11 Data Quality Control. After raw data of the second-generation sequencing was obtained, the quality of the original reads was examined using the FastQC online tool (http://www.bioinformatics.…”

“…The experiment was completed in the preparation of the entire library. The constructed library was sequenced with an Illumina Hiseq 2500 at a sequence read length of 1 × 36 bp …”

Sensory Ecology of Predator-Induced Phenotypic Plasticity

“…are becoming available (Kato et al, 2012; Nakanishi et al, 2014; Naitou et al, 2015; Nong et al, 2017). Many studies have investigated the changes in gene expression levels that affect morphological defense expression in Daphnia pulex (Schwarzenberger et al, 2009; Spanier et al, 2010; Miyakawa et al, 2015; Rozenberg et al, 2015, 2016; Hales et al, 2017), while only a few have looked at the (neuro-) physiological changes (Hanazato, 1991; Barry, 1999, 2002; Weiss et al, 2012a; Miyakawa et al, 2015).…”