De novo assembly and annotation of transcriptomes
Highlights

De novo assembly or annotation
of unknown transcriptomes

Different library types for each
application assuring optimal
performance

Normalisation significantly
reduces sequencing effort
Introduction
Even though many genomes have been sequenced so far, the
decryption of a transcriptome gives additional insights into the
biology of an organism. RNA sequencing can be used to reliably
assemble and annotate sense and even antisense (see strandspecific library) transcripts of an unknown transcriptome.
Specialised workflows meeting all needs
Strand-specific library
Prokaryotes and eukaryotes with
small genomes have a
transcriptome with a high density,
i.e. transcripts are very closely
packed and can even overlap.
Moreover, some parts of the
genome can be transcribed in both
directions giving rise to sense and
anti-sense transcripts.
To address this complexity, we
established our strand-specific
library protocol. This library type
retains directional information
allowing easier verification of
transcripts.. In addition, it allows
for the discrimination of sense and
anti-sense transcripts which are
present even in higher eukaryotes.
The input material is high quality total RNA from tissues or cells and
can be derived from eukaryotic or prokaryotic organisms. Total RNA
contains high amounts of ribosomal RNA (rRNA). This is especially
true for prokaryotes in which up to 95% of total RNA is rRNA.
Prokaryotic rRNA will be removed by rRNA depletion. As ribo
depletion is optimised for most standard organisms, please contact
us to check the availability for your organism of interest.
Eukaryotic mRNA is enriched by poly(A) selection. Subsequently, the
enriched mRNA is reverse transcribed into cDNA using random
hexamer primers and then normalised to reduce the relative amount
of highly expressed transcripts (e.g. house-keeping genes). Expression
levels of high and low abundant transcripts can still differ even after
normalisation, but the differences in frequencies will be reduced by
several orders of magnitude.
Sequencing is performed using the Illumina MiSeq platform to
generate millions of long reads which are used to generate a de
novo assembled transcriptome or to annotate the transcriptome
with the help of a reference genome.
Further reading on RNA sequencing
Ozsolak, F., & Milos, P. M. (2010). RNA sequencing: advances,
challenges and opportunities. Nature Reviews Genetics, 12(2),
87–98. doi:10.1038/nrg293
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Germany, March 3, 2014-3