Data pre-processing
- Quality filtering
- Adapter trimming
- Discarding rRNA-mapped reads or contaminants (per request)
Output files include: quality report (HTML or PDF formats), filtered reads in FASTQ format (per request)
Fig. 1. A detailed and self-explanatory quality report for each sample provided.
Expression analysis
- Estimating gene and transcript expression values
- Differential expression analysis
- Enrichment analysis of KEGG pathways and Gene Ontology terms
- Output files include:
- Tab-separated files with calculated expression values for genes and transcripts; these files might be open in a spreadsheet, such as MS Excel. The expression values are provided in FPKM/RPKM and TPM units
- A list of differentially expressed genes, with fold change values and adjusted P-values
- Raw expression values for each gene in each condition as a single table (per request)
- A heatmap of differentially expressed genes in PDF format
- MA and volcano plots - essential diagnostic plots for differential expression analysis, in PDF format
Fig. 2. MA plot and Volcano plot, essential diagnostic plots for differential expression analysis.
Ab initio transcriptome assembly (genome dependent)
- Read mapping against a reference genome sequence
- Quality control of mapping results and post-processing them
- Ab initio assembly of the transcriptome (detection of novel genes and splicing isoforms, including antisense and intronic transcripts)
- Quality filtering of the transcriptome (if required)
- Comparison with a reference transcriptome/annotations (if available)
- Possible further steps: Expression analysis; Annotation of transcriptomes
- Output files include:
- Quality report from read mapping
- A file in BAM format containing raw results of read mapping (per request)
- The transcriptome in GFF/GTF and FASTA formats; the GFF/GTF files might be used for direct visualization of the transcriptome in genome browsers, such as IGV
- Sashimi plots for genes of interest (per request)
Fig. 3. Read mapping results in BAM format
Fig. 4. Sashimi plot
De novo transcriptome assembly (does not require genome sequence)
- De novo assembly of the transcriptome
- Calculation of quality measures
- Possible further steps: Expression analysis; Annotation of transcriptomes
- Output files include:
- a transcriptome in FASTA format
- quality report for the transcriptome assembly
Annotation of transcriptomes
- Identification of protein-coding genes
- Annotation of proteins: finding structural and functional domains; similarity search against databases of annotated proteins in other organisms
- Assignment of KEGG pathways and Gene Ontology (GO) terms
- Identification of tRNAs, rRNAs, snoRNAs, snRNAs
- Identification of long non-coding RNAs (lncRNAs)
- Identification of circRNAs
- Possible further steps: identification of microRNAs
- Output files include:
- A single tab-delimited file (to be open in a spreadsheet program, such as MS Excel) with most of annotation results, including assigned KEGG and GO terms and found protein domains
- FASTA and GTF/GFF/BED files with identified open reading frames and different classes of non-coding RNAs
- Interested in other types of output files? Feel free to ask us
Examples of other, RNA-Seq related types of analysis:
- Finding RNA editing events
- Finding targets for microRNAs
- Analysis of splicing (different variants)
- Output files include:
- A single tab-delimited file (to be open in a spreadsheet program, such as MS Excel) with most of annotation results, including assigned KEGG and GO terms and found protein domains
- FASTA and GTF/GFF/BED files with identified open reading frames and different classes of non-coding RNAs
- Interested in other types of output files? Feel free to ask us
