Plasmid sequencing

Samples must be sent to e-Zyvec SAS, NGS, 80 rue du Dr Yersin, 59120 Loos – FRANCE.

To ensure a good process, please send between 10-25µL volume of your template (≥50ng/µL) witch means at least 500ng.

If your samples are less than the amounts indicated let us know we can provided further assistance. Samples must be in a Nuclease-free H2O or 10 mM Tris-HCl pH 8.0 solution. The reference purity is to have an OD A260/A280 ratio ≥ 1.8 and A260/230 ≥ 1.9.

We have developped our inhouse analytic pipeline. Based on NGS raw data obtained using Illumina technology, we combine

We operate routine sequencing of purified plasmids whatever their size (so far, we went up to 23Kb). However, using our ISeq100, we can also analyze other DNA molecules such as PCR amplicons, Cosmids or Bacmids, RNAs (RNAseq), or genomic DNA. Such projects will be undertaken as ‘tailored-projects’, so please, contact us for feasibility assessment and quotes.

A PDF report will be sent by e-mail (you can also download the results in your project management page on www.e-zyvec.fr) to you summarizing the analysis of your sequences, in addition to this report, we provide various files:

. If the results are consistent enough with the expected sequence of a given vector, we provide you the corrected sequence (_corrected.fasta) highlighting the differences found.

. If the results are not consistent enough with the expected sequence (for example large insertions or deletions), or if an expected sequence was not communicated, we provide you a de novo assembly file (_assembly.fasta). Note that there may be several sequences (contigs) in this file in case of fragmented assembly, and that you may have to further analyze these results.

Incomplete assembly may occur for various reasons:

1- In case there are long (>300bP) repeated sequences in your molecule (promoter duplication for example) NGS analysis will not be able to confirm that all duplications are effectively present. In such case we may operate complementary analysis upon to confirm duplications, upon demand, or advise you on how to proceed for such validation by yourself.

2- Extreme High or Low GC content may result in sub-optimal reading with Illumina technology (as well as for any known technology). If such difficult sequences stretch over long (>300bp) portions of your molecule this may impair the analysis locally.

3- Mixed samples (contamination of your vector with another vector or too much genomic DNA) will result in several contigs that cannot be discriminated.

On top of the technical quality control recommended by Illumina and reagents manufacturer, we have included some analysis controls in our pipeline. One of these is the ratio of reads unmapped to the expected sequence.

If too high it would indicate a sample contamination by either another plasmid or some genomic DNA, or even the presence of unexpected material inserted within your vector.In such case we will provide you the corresponding contigs assembled from the unmapped data (if any relevant).

The regular service is based on a unit price for each reaction, and the estimated time is 2 to 3 weeks. Basically, your samples are queuing until the next available run (so it might be quicker than the estimated times if you’re lucky).

The express service time is estimated at 3 days, this service being available for orders up to 12, 24, 48, 72 and 96 reactions. In this case we are dedicating a full run to your analysis, hence the short delay.

These services do not differ on the sequencing methodology employed, or the analytic pipeline for plasmids validation.