Description
Material provided: Data (raw sequence files) and Bioinformatics analysis
Unit definition: 1x RNA sample from living or fixed cells or organs
Global Transcriptome determination and differential expression analysis is often hampered by the availability of very small amounts of starting material from various biological sources. Total RNAs prepared from small amounts of tissues or cells is often degraded, which in conjunction of the low amounts (eg. < 50 ng total RNA) makes them inappropriate for use in classical RNA-sequencing methods. For such cases, we offer 3’-end sequencing assays, which can offer highly reproducible, quantitative transcriptome determination. More precisely, according to the amounts and the quality of the total RNA provided by the user we implement 3 protocols with modifications: (a) Standard protocol for > 10 ng input material; (b) Low input protocol A for 1 to 10 ng of RNA prepared from low quality/degraded RNA; (c) Low input protocol B for ≤ 1 ng total RNA
The service offered uses Lexogen’s 3’Quant-seq method (Lexogen), where library preparation is initiated by oligo-dT priming and therefore no prior poly-A enrichment of mRNA or ribosomal RNA depletion is required. First-strand synthesis and RNA removal is followed by random-primed synthesis of the complementary strand. Illumina-specific linker sequences are introduced by the primers. The resulting double-stranded cDNA is purified with magnetic beads. Library PCR amplification then introduces the complete sequences required for cluster generation. The complete workflow is explained in the link below: https://www.lexogen.com/quantseq-3mrna-sequencing/#quantseqworkflow
The Lexogen Quantseq Forward kit is used, which generate Illumina compatible libraries of sequences close to the 3’end of the polyadenylated mRNA. Only one fragment per transcript is generated, which makes the protocol more accurate for quantitative assessment of even low copy transcripts. The expected yield is 5-10 million reads per sample of 75 nt single read sequence output per RNA sample from Illumina Next-Seq 500 platform. Sequence reads are subjected to:
Quality Control of the input data; Pre-processing of the reads; Mapping to reference genome; Transcriptome assembly and identification of differentially expressed genes; Downstream data analyses.
The service also includes bioinformatics analysis. Data processing and analyses are performed by PANDORA (Moulos and Hatzis 2014 doi: 10.1093/nar/gku1273 ; and Fanidis and Moulos 2020, doi: 10.1093/bib/bbaa156), implemented in MetaseqR2 Bioconductor Package, which interfaces several normalization and statistical analysis algorithms and generates a fully interactive report and genome browser visualizations, more efficiently reflecting true experimental outcomes. For each Experiment we provide the raw data in fastq file format and a complete MetaseqR2 Report, which includes:
- Analysis Summary
- Figures: Multidimensional Scaling; Biotype detection; Biotype detection counts; Read and Biotype saturation; RNA-seq reads noise; Correlation plots; Pairwise scatterplots; Boxplots of unnormalized and normalized data; RNA composition graphs; GC content bias; Gene/transcript length bias; Chromosome and biotype distribution of filtered genes; Volcano plots; Differential Expression Heatmaps; Chromosome and Biotype distributions of Differentially Expressed Genes
- Tables: Excel File of differentially expressed genes and read statistics.
For more information, please contact us.
IMBB Technical Support:
Email: bioinfo@imbb.forth.gr
Telephone: 0030 2810 391159