Tag Archives: de novo sequencing

Hybrid De Novo Genome Assemblies

What are your intentions when being interested in a bacterial or fungal de novo genome sequencing project?

Typical answers we get from our customers:

  • Easy working with the data
  • Data suitable for high quality annotation
  • Resolution of structural rearrangements
  • High consensus accuracy
  • High cost-efficiency

All these requirements can be fulfilled perfectly when combining Roche GS FLX++ and Illumina data. The long Roche FLX++ reads of up to 1100 bp give much longer contigs than Illumina reads only do. For scaffolding and to be able to resolve structural rearrangements we sequence shotgun (SG) and LJD libraries with Illumina technology. The adding of Illumina reads keeps the overall costs at a reasonable level. Furthermore the reads correct the Roche sequencing errors at homopolymer sites and therefore enable us to build a consensus sequence with high accuracy.

The superiority of such a hybrid assembly becomes quickly apparent when looking at the following results of one of our proof of concept studies. In this de novo project, we sequenced a fungal genome of about 30 Mbp and approx. 57% GC content. Using the hybrid strategy we obtained only 10 chromosome-sized scaffolds (see figure below) with up to 8.3 Mbp. Remarkably, the 10 scaffolds represent the majority of genetic information present, given that they make up 99.6% of all scaffold sequence information.

Such results enable easy data handling and definitely are an excellent starting point for annotation and studying of gene content and rearrangements.

Sequencing strategy: SG library with FLX++ (approx. 10-fold coverage), SG and LJD 3 kbp, 8 kbp and 20 kbp on Illumina HiSeq 2000 with 2x 100 bp module.

 

MiSeq – soon in its full bloom?

Rather than resting on the successes of the MiSeq launch, Illumina is continuously improving the performance of their small Benchtop Next Generation Sequencing System. Geoff Smith is talking about improvements in the read lenght of the MiSeq in the video attached.

This instrument is not only another next gen sequencing device but really has remarkable advantages over other instruments when sequencing for example bacterial genomes. This is why I am really delighted that we now can offer services using the MiSeq instrument. (More info on our MiSeq services can be found here)