Efficient strategies for de novo sequencing of small, mid-size and large eukaryotic genomes
Obtain high quality genome de novo assemblies by sequencing a combination of shotgun libraries long jumping distance libraries.
Eurofins Genomics offers libraries with wide jumping distances to easily tackle any genome size - from bacterial genomes to large and complex eukaryotic genomes.
- Long jumping distance (LJD) libraries to be sequenced on Illumina HiSeq 2500 / MiSeq
- LJD libraries are available with jumping distances of 3 kbp, 8 kbp, 20 kbp and 40 kbp
The LJD fragments comprise of 2 DNA fragments originally 3 kbp, 8 kbp, 20 kbp or 40 kbp apart in the genome of interest. The long paired-end reads are used to determine the orientation and relative position of the contigs generated during the data assembly of shotgun reads. With Eurofins Genomics unique library portfolio, spanning gaps or repeats in your genome of up to 40 kbp is achieved in a fast and efficient manner.
Working closely together with our customers, we perform de novo genome sequencing projects in the following stages:
- Ultra high throughput sequencing of a shotgun library and assembly into contigs
- Scaffolding of contigs by long paired-end sequencing
- Optional, closing of gaps based on Sanger technology
- Optional, bioinformatic analysis, e.g. annotation or comparison of different organisms
Get the longest contigs and scaffolds
Reduce your post-sequencing efforts to a minimum.
Since we first started offering our NGS services with the Roche 454 GS20 system in 2006, Eurofins Genomics is highly adapted in de novo genome sequencing. We have sequenced over 100 microbial and fungal genomes with our special de novo strategy, such as E. coli, Streptomyces, Chlamydomonas, Lactococcus, Clostridium, Mycoplasma, Pseudomonas, Fungi, etc.
The gold standard for de novo sequencing of small genomes combines backbone sequencing (shotgun-libraries) and scaffolding using paired-end libraries with large jumping distances. By strategically combining sequencing and scaffolding of up to 5 libraries we get the longest average scaffold sizes and smallest number of remaining gaps. This strategy drastically reduces your post-sequencing efforts by cutting down on gap closing and manual editing time.
The appropriate layout of different LJD libraries as well as the sequencing technology depends mainly on the complexity & size of your genome. We would be happy to advise you to find the most convenient strategy:
Types of paired-end libraries
Combine paired-end libraries with a shotgun library and bridge gaps up to 40 kbp. Choose from paired-end libraries with insert sizes of 3, 8, 20 and 40 kbp.
Our recommendations at a glance:
|Small fungi and bacterial genomes
||Shotgun + 8 kbp library
|Large fungal genomes
(Shotgun + 3 kbp + 8 kbp + optional 20 kbp)
Sequencing of paired-end libraries
Scaffolding can be performed on HiSeq 2500 and MiSeq. LJD libraries are available with jumping distances up to 40 kbp.
Size does not matter any more
Rely on the comprehensive experience of Eurofins Genomics for de novo sequencing of large eukaryotes.
With prices / bp dropping eukaryotic genome sequencing projects are rapidly being implemented in projects worldwide. Eurofins Genomics has gained profound experience in de novo sequencing and assembly of the larger, more complex, and repeat-rich genomes of higher eukaryotes. Our expertise ranges from insects to large plants with up to 8 Gbp and from Roche GS FLX only strategies to projects where backbone sequencing and scaffolding has been performed with the Illumina HiSeq 2000.
One of the big challenges of such projects is the scaffolding of the contigs from shotgun sequencing. That is why Eurofins Genomics focused on developing paired-end libraries with large jumping distances for sequencing by the Illumina HiSeq 2500. These unique long jumping distance (LJD) libraries offer ultra-high throughput and cost-efficient scaffolding of contigs.
The LJD libraries are different from Illumina’s mate-pair protocol since they allow adaptor-based ligation to offer higher assembly accuracies.
The appropriate layout of different LJD libraries as well as the sequencing technology depends mainly on the complexity & size of the eukaryotic genome. We would be happy to advise you about the most convenient strategy.
- The type of long jumping distance (LJD) libraries combined with the shotgun library. Choose from libraries with insert sizes of 3 kbp, 8 kbp, 20 kbp and 40 kbp and bridge gaps of up to 40 kbp.
- Optional, include cDNA library sequencing in order to support gene annotation or take advantage of sequencing specialised libraries, like for example methylation filtered libraries that support coverage of transcriptional active euchromatic parts of the genome.
Need more information for your specific project or an individual quote? Just contact us!
BACs, fosmids, plasmids, and viruses
Sequence your small genomes and large insert constructs in a cost-efficient and advanced way irrespective of the type.
Achieve high quality de novo sequencing of small genomes and constructs by sequencing your sample using a long read technology (PacBio / Roche 454). Easily bridge small repetitive elements and benefit from longer contigs.
The successful completion of more than 2000 BAC projects built our expertise in sequencing all kinds of small genomes and insert constructs like:
- BACs, PACs, cosmids and fosmids
- Plasmids and phages
- Mitochondria and chloroplast samples
- DNA- and RNA-viruses
In projects where only small DNA amounts are present and a circular genome is used, we also offer reliable amplification of sample material using a rolling circle polymerase.
Long jumping distance libraries
Achieve high throughput scaffolding using our proprietary long jumping distance (LJD) libraries.
Based on our long-term expertise with long paired-end libraries, we developed LJD libraries adapted to the Illumina HiSeq 2500 technology.
LJD libraries harbour several advantages over the Illumina mate-pair libraries since they contain a spacer to tag the crossover point between both fragments (see figure in sidebar). Find a list of the advantages below.
Span large repeats in your genome
We offer LJD libraries with 3 kbp, 8 kbp, 20 kbp and even 40 kbp jumping distances to resolve large and complex repetitive structures.
The Illumina mate-pair library is available with a jumping distance of only 2 kbp to 5 kbp.
No misassembly with hybrid reads
Our LJD libraries have a built in spacer to prevent misassemblies when changeover occurs within the (hybrid) read. The spacer defines the exact location where a changeover occurs thus offering a physical separation between both fragment ends.
The Illumina mate-pair protocol and other mate-pair protocols built on it do not offer a physical separation between the two fragment ends. Therefore, sequencing of a mate-pair library will generate a huge quantity of hybrid reads that contain sequence information from one and the other end of the original fragment. These reads can lead to severe misassemblies.
LJD reads with proven insert size
Adjustments in the LJD pull-down protocol highly reduces the number of shotgun paired-end reads. With LJD libraries only about 1% of reads are shotgun read pairs.
Shotgun like paired-end reads can occur when not all unbiotinylated fragments are washed away in the library preparation procedure (see figure in sidebar). These read pairs have a gap size of only 300-500 bp. They do not contribute to bridge large repeats and the wrong classification can lead to misassemblies. Contamination rate of Illumina mate-pair reads with shotgun read pairs is usually about 2-5%, sometimes up to 30%.
If you have further questions about our libraries or need specific information for your project, just contact us!