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Metatranscriptomics / Transcriptomics

RNA sequencing (RNA-seq) using NGS enables the identification and quantification of gene expression in biological samples. DNASense offers RNA-seq of individual prokaryotic and eukaryotic organisms (transcriptomics) as well as mixed microbial communities (metatranscriptomics). We provide full sample-to-answer RNA-seq solutions with cutting-edge bioinformatic pipelines for identification of differentially expressed genes (DEGs). Our workflows support mapping and identification of RNA transcripts by either de-novo assembly, reference genomes or purpose built reference-hybrids (please refer to our genomics/metagenomics ).

A crucial prerequisite for useful RNA-seq results is a suitably scaled experiment design. The number of biological replicates and required sequencing depths are highly dependent on the experiment’s scientific purpose, sample complexity and total transcript content. For large-scale projects setting up a pilot study is always the recommendation. The table below provides general recommendations, but please contact our specialist for further information.

Strategy Example sample environment mil reads/sample List price/sample
12 samples 24 samples
Transcriptome Prokaryote 5 900 650
Eukaryote 25 1120 837
Metatranscriptome Complex microbial community 50 1320 1039
Strategy Example sample environment mil reads/sample List price/sample
12 samples 24 samples
Transcriptome Prokaryote 5 6700 4850
Eukaryote 25 8325 6225
Metatranscriptome Complex microbial community 50 9800 7725

Our standard package includes: pre- and (optional) post-project meeting with a DNASense specialist, RNA extraction, rRNA depletion, library preparation and cDNA sequencing, DEGs analysis (mapping/de novo assembly, gene annotation, quantification and statistical analysis), online-access to raw data and result files, a detailed project report with ready to use for publication materials and methods and publication grade illustrations.

Add-on services (non-exhaustive list): functional annotation (KO, GO), functional enrichment analysis, LEfSe analysis, manual curation of metabolic pathways, Data submission (e.g. ENA).

FAQ

  • Ribosomal RNA constitutes up > 80% of cellular RNA.  Our recommendation is to perform rRNA depletion for RNA high yield samples, to maximize focus RNA in the subsequent sequencing. For RNA low-yield, for which RNA-depletion due to recovery loss is not a recommended option, increasing sequencing depth may be used as an alternative solution.

  • Statistical power increases with increasing number of replicates, especially for low-abundant genes. Our recommendation is a minimum 4 biological replicates per condition.

  • Depends on the scientific question at hand and sample matrix. For DEGs 2-5 million mapped reads for pure cultures is sufficient for most prokaryotes, for whole transcriptome sequencing 100-200 mio reads is typical.

  • The transcriptome/metatranscriptome is prone to mRNA degradation and changes in expression profile. Hence, to minimize biases associated with sampling, special care must be taken. We recommend adding a mRNA preservative (preferably RNAlater) and snap freezing samples as soon as possible.

Mie Bech Lukassen, Ph.D.
Chief Operating Officer

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