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Transcriptome Sequencing

The GS FLX and GS Junior Systems offer the powerful combination of long sequencing reads and dedicated GS Assembler software, enabling sequencing, de novo assembly and analysis of messenger RNAs (mRNAs) that represent the comprehensive transcriptome of an organism.

  • De novo transcriptome assembly – Long reads enable gene annotation of previously uncharacterized or poorly characterized genomes. An attractive alternative to whole genome sequencing – requiring fewer sequencing runs to obtain a comprehensive picture of the functional transcribed elements of the genome.
  • Discover novel transcripts and isoforms – Long reads enable detection of alternate gene splice isoforms, fusion transcripts, and translocations.
  • Identify the full range of expression - Detect nucleotide polymorphisms in the transcribed portions of the genomes and allele-specific expression.
  • New! GS FLX+ reads up to 1,000 bp - Cover more exons and splice junctions with single reads and extend further into untranslated regions. Improve long transcripts coverage for more accurate reconstruction of gene models.

Using the system's long reads and the GS De Novo Assembly, 12 full length Drosophila PTEN gene variants were fully reconstructed for this sample, including 5 of the 6 known variants, with internal splice junctions matching perfectly to the reference, and 7 novel variants. Assembly of simulated 100 bp short reads generated two partially reconstructed and fragmented transcript variants. These short read assemblies contain ambiguous splice junctions that did not agree with the reference - attributed to their inability to span across enough exons to create unique aligned isotigs.

454 Sequencing Systems offer optimized protocols for sequencing of samples with as little as 500 pg total RNA.

cDNA Rapid Library Prep

While many suitable methods exist for the preparation of cDNA for sequencing, The GS FLX and Junior Titanium cDNA Rapid Library Preparation method relies on random hexamer synthesis of cDNA. Using 200 ng of messenger RNA as starting material, a complement DNA (cDNA) library can be quickly prepared using the Roche kits: Primer Random, cDNA Synthesis System and the GS Rapid Library Prep Kit.

Low-Input RNA Samples

The use of NuGEN’s Ovation® RNA-Seq System with the GS FLX and GS Junior Titanium cDNA Rapid Library Prep protocol enables whole transcriptome analysis of samples with as little as 500 pg of total RNA. > Learn more

GS De Novo Assembler - Transcriptome Assembly Tool

  • Dedicated GS Assembler software is designed specifically for de novo assembly of transcripts, supporting the particular needs and conditions of transcriptome analysis.
  • Easy-to-use GUI and command line user interfaces enable straightforward analysis for both biologists and bioinformatics users.
  • Automatically align sequencing reads into isoforms ready for uploading into publicly available data analysis and visualization tools.

One step discovery and annotation. Figure shows an example of the de novo assembly of cDNA reads and their alignment to the corresponding fungal gene. The long Expressed Sequence Tags (EST) reads (green) provided by the GS FLX Titanium chemistry routinely span multiple exons - shown bridging up to 9 of the 19 exons in this example. The GS De Novo Assembler produced two full-length EST contigs (blue), revealing two full-length variants with alternative splicing occurring in the 5' untranslated region (UTR). While no reference sequence was available for this newly sequenced species, the GenMark predicted gene is shown (red). Screenshot generated with the Argo genome browser.

Useful URLs on Automated Annotation Systems

  1. NCBI non-redundant Clusters of Orthologs, COG: http://www.ncbi.nlm.nih.gov/COG/
  2. Kyoto Encyclopedia of Genes and Genomes, KEGG: http://www.genome.jp/kegg/
  3. MEGAN: http://www-ab.informatik.uni-tuebingen.de/software/megan/welcome.html
  4. CARMA: http://www.cebitec.uni-bielefeld.de/brf/carma/carma.html
  5. Marine Microbial Ecology: CAMERA: http://camera.calit2.net
  6. National Microbial Pathogen Data Resource (NMPDR): http://rast.nmpdr.org/
  7. Rapid Annotation using Subsystems Technology (RAST) server, which is viewable through the SEED-viewer
  8. National Center for Biotechnology Information’s BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi
  9. RSAT’s Pattern Discovery Tool: http://rsat.ulb.ac.be/rsat/
  10. EMBOSS’s palindrome: http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome
  11. Open source expandable software for the microbial ecology community, MOTHUR, DOTHUR, http://www.mothur.org/