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Frequently Asked Questions About Our Products And Services

How much sample material do you need?

2-5 µg is the required amount of genomic DNA for shotgun sequencing on Illumina HiSeq 2500 / MiSeq.

Additional sample material is needed for the preparation and sequencing of LJD libraries with jumping distances of 3 kb, 8 kb, 20 kb and 40 kb. In this case, 5 µg, 15 µg, 30 µg and 50 µg respectively of extra DNA should be provided.

If possible, we would encourage you to submit some more sample material to allow for more intensive QC and repetition in case of failure.

What does coverage sequencing mean?

This involves sequencing of a DNA until a certain number of sequence information and therefore sequence coverage is reached.

Example: A 1-fold sequence coverage of a 3 Mb genome in size means 3 million base pairs are determined, or a 6-fold coverage means 18 million base pairs are determined.

For genome sequencing using the Illumina technology we recommend a coverage of 30-50-fold.

What is the delivery time?

It depends on the organism and your requirement. For the coverage sequencing of a bacterial genome like E. coli (4.6 Mb), the delivery time is just a few weeks.

How do you deliver the data?

Depending on the amount of data, we ship data DVDs, USB sticks, or hard disk drives.

In addition to that we offer download of the data from our secure FTP server.

How is the assembly and scaffolding process done?

The assembly process assembles single overlapping reads to larger contigs. In the first instance, the contigs are not ordered. Scaffolding means to determine the order and orientation of the contigs. It can be achieved for example by sequencing long jumping distance libraries on the Illumina instruments. This technology creates pairs of short sequence tags, which are originally positioned 3 kbp, 8 kbp, 20 kbp or 40 kbp apart from each other.

Scaffolding matches the paired ends to the contigs under consideration of the original distance and thereby brings the contigs into order.

What is a mate-pair library?

Mate-Pair library consists of approximately 150-300 bp fragments. They are composed of 2 DNA segments originally located 2-5 kb apart in the genome of interest. With a mate-pair library it is therefore possible to span gaps or repeats of up to 2-5 kb.

For library preparation genomic fragments are sheared and size selected for approx. 3.5 kbp fragments. The fragments are end labelled with biotin and circularised. The circularised molecules are sheared once more and the library is enriched for biotin labelled fragments. Sequencing these biotinylated fragments generates the desired 'outward-facing' paired reads, meaning they align to a reference sequence outward-facing from each other. These outward-facing reads align to the reference sequence with a gap size of 2-5 kbp.

The mate pair library has several restrictions, limiting the usefulness of the library for high quality scaffolding of contigs:

1) Inward facing reads: Unbiotinylated fragments that have not been successfully washed away will cause undesired inward facing reads (see Illumina’s mate pair sample preparation guide). These inward-facing reads are like shotgun paired end reads as they align to the reference with a smaller gap size of around 200-300 bp.

2) Genomic shearing of the circularized biotinylated molecules is a mechanically process and will produce fragments having the biotin label in the middle of the fragment, but also on all other positions of the fragment. When sequencing fragments having the biotin label (and thus the crossover) in the middle of the fragment the desired outward-facing reads will be generated. However, when sequencing fragments having the biotin and the crossover at the outer 100 bps of the fragment, one of the resulting reads will be a hybrid read. The hybrid read will contain sequence from one AND the other end of the original 3.5 kbp fragment and there is no information about the location of the changeover. These read pairs are worthless for the assembly and can even lead to misassemblies.

For high quality high-throughput scaffolding by Illumina sequencing Eurofins Genomics offers an alternative proprietary library, the so called long jumping distance libraries (LJD's) with jumping distances of 3 kbp, 8 kbp, 20 kbp or 40 kbp. Read more

What is a long jumping distance (LJD) library?

Eurofins Genomics has developed long jumping distance (LJD) libraries for high throughput scaffolding of contigs. The LJD library is adapted to the Illumina HiSeq 2000 or the Illumina MiSeq technology. While the mate-pair library displays jumping distances of 2 kbp to 5 kbp, LJD libraries do show much more precise distances, thus improving assembly quality. LJD libraries are being offered for 3kbp, 8 kbp, 20 kbp and even 40 kbp jumping distance.

In addition, other advantages argue for the LJD library:

1. Because of the differences in library generation the number of shotgun paired end reads is greatly reduced. Only about 1% of LJDs reads are shotgun paired-end reads.

2. Within the LJD library the sequences from one and the other end of the original fragment (3 kbp, 8 kbp, 20kbp or 40 kbp) are separated by an adaptor sequence. Hybrid reads, as present in mate-pair libraries (see explanation in the mate-pair FAQ) are therefore almost completely eliminated when using LJD libraries. The changeover is defined by the presence of the adaptor.

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