A platform independent RNA-Seq protocol for the detection of transcriptome complexity

Abstract

BackgroundRecent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform.ResultsWe set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol.ConclusionWe tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-855) contains supplementary material, which is available to authorized users.

De novo transcriptome sequencing and analysis of male and female swimming crab (Portunus trituberculatus) reproductive systems during mating embrace (stage II)

“…A number of studies have analyzed the gonad transcriptome and provided information about the molecular mechanisms underlying gonadal development and maturation [ 15 – 17 ]; but few have focused on the reproductive system. Various sequencing platforms have also been established, such as Solexa illumine, Roche 454, and ABI solid [ 18 – 20 ]. These technologies have been applied in studying the transcriptome of many aquatic animals, particularly in crustaceans [ 18 – 20 ].…”

“…Various sequencing platforms have also been established, such as Solexa illumine, Roche 454, and ABI solid [ 18 – 20 ]. These technologies have been applied in studying the transcriptome of many aquatic animals, particularly in crustaceans [ 18 – 20 ].…”

Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode

“…These sequences were then used as controls in the following experimental and analysis steps. Furthermore, MPE4 and MPE5 samples were prepared for pyrosequencing adapting the sequencing library preparation protocol described by Calabrese et al (2013) . The libraries were then deposited on 2/8-lane PicoTiterPlate (PTP) wells (Roche/454; Roche, Basel, Switzerland) and sequenced in both directions on GS FLX Titanium pyrosequencing platform.…”

“…We performed a standard read rejecting and trimming procedure, using GS Run Processor V2.4 (Roche 454 Life Sciences software package; Roche, Basel, Switzerland), with the parameters suggested by the 454 platform manufacturer. Due to amplicons ligation and subsequent nebulization steps carried out for sequencing library preparation (see Calabrese et al, 2013 ), we executed a primer search analysis ( Supplemental Information 1 ) generating forward and reverse reads data sets, which were processed separately in the subsequent analyses. Pyrosequencing and PCR noises were removed by invoking PyroNoise and SeqNoise algorithms respectively, available from AmpliconNoise package ( Quince et al, 2011 ), with their default parameters.…”

Transcriptome Analysis on RNA-seq Data

“…In past decade, RNA-seq is emerged as one of the most versatile application of Next-generation Genome Sequencing (NGS) technology and revolutionized the global researches on transcriptome [1]. The high-throughput RNA-seq data continues to provide unparalleled insight into transcriptome complexity [2]. Now one can consider that the "gold standard" for assessing global transcriptome analysis on RNAseq data is poised to revolutionize our understanding of transcription and posttranscriptional regulation of RNA [3].…”