Customized strategies to specifically answer your scientific question

Study transcriptional activity in any organism without reference

Your transcriptome project

Full length transcriptome de novo sequencing enables the detection of novel and rare transcripts and the accurate characterization of splice variants and fusion transcripts. Furthermore the data can be used to improve genome assemblies and gene annotation of so far uncharacterized genomes.

Strategy

To perform a most accurate de novo assembly and hence enable a more complete reconstruction of full length transcripts long sequencing reads are crucial. A single read can completely cover whole transcripts, exons and splice junctions and therefore allows the identification of alternate isoforms. Further enhance the gene discovery rate and reduce complexity through preparation and sequencing of normalized cDNA libraries.

Starting material

• tissue
• total RNA
• mRNA
• cDNA

Library preparation

• Optional: mRNA enrichment / rRNA depletion for prokaryotes
• Oligo(dT) or random primed cDNA synthesis
• Normalization through denaturation / reassociation of cDNA

 Sequencing

• Roche GS FLX+ or PacBio RS I single reads
• Improvement of sequence quality through additional Illumina HiSeq 2000 data

Bioinformatics

• Raw data (fasta and fastq)
• Flowgrams (sff)
• Clustering using cd-hit-est
  • Representative of each cluster (fasta)
  • Clustering table (tsv)
• Semiautomatic Assembly using GS de novo assembler
  • Singletons and assembled contig sequences (fasta)
  • De novo assembly report (txt)
  • Assembled contigs coverage report (txt)
• Blastn w/ GenBank or customer databases
  • Cluster / EST contigs annotation table (tsv)

• Pre-Sequencing
• Single Reads
• Genomes
• Targeted (re)-sequencing
• Human Sample Sequencing
• Transcriptomes
  • > De novo Sequencing
  • > Re-Sequencing
• Metagenome
• Regulome

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