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Instructions pour la préparation des échantillons

Please read carefully these guidelines before preparing your material for sequencing

Samples should be in 1.5mL or 2mL tubes.

Write distinctly the sample codes received with your quote on the lid of your tubes.
No stickers please.

Feel free to add any useful information on the side of the tube.

We exclusively rely on the Qubit fluorometric quantitation system (Thermo Fisher) for any nucleic acid quantification. We will perform a Qubit quantification of your samples shortly after reception. Note that spectrometric methods (such as Nanodrop) often provide an overestimation of the concentration and are very sensitive to contaminants (chemicals or other nucleic acids).

If your material does not follow the described criteria, please contact us before sending your samples.

Genomic DNA libraries :

Most kits provided by biotech companies usually work for sequencing purpose. There are no particular precautions that should be taken apart from usual molecular biology precautions.

DNA should be checked by running an aliquot on agarose gel. There should be no significant degradation.

DNA should be in a clear solution without any precipitate at room temperature.

  • For Truseq protocol : 200-2000ng in 100µL volume max. DNA should be resuspended in Tris 10mM buffer. This protocol allows for a more precise insert fragment size. Being in the higher range quantity allows to perform fewer PCR cycles leading to higher quality libraries. If necessary, this protocol can be used to construct libraries from partially degraded DNA. However, in these cases, be aware that sequencing results might not be optimal.

  • For Nextera protocol : 50-100 ng of high quality DNA (no or very low degradation visible on agarose gel). The 260/280 ration should be 1.8. DNA has to be in buffer WITHOUT EDTA (lots of kits provide TE buffer ; use Tris-HCl 10mM or H20 instead).

  • For NexteraXT protocol : (for bacterial genomes only) : 5-10 ng of high quality DNA (no or very low degradation visible on agarose gel). The 260/280 ration should be 1.8. DNA has to be in buffer WITHOUT EDTA (lots of kits provide TE buffer ; use Tris-HCl 10mM or H20 instead).

    • Genomic Mate-Pair libraries : 10 µg of DNA in H20 or 10mM Tris HCl buffer.

ChIP-Seq libraries :

> 2 ng of 100-400bp dsDNA. If your fragments are not in this size range please ask us for a protocol modification. Libraries made from <5 ng DNA might not be of optimal quality, especially if outside of the optimal size range.
Libraries with lower amount of DNA can be constructed, however we cannot guarantee their quality in such conditions.

RNA-seq libraries :

Most commercially available RNA extraction kits are compatible with downstream NGS analyses ; it is often mentioned in the documentation if a kit is compatible with RNA-seq analysis. All RNAs have to be in nuclease-free H2O.

RNA-seq library preparation is very sensitive to genomic DNA contamination especially when the samples are treated for rRNA depletion (Ribo-Zero). This is less critical for libraries with polyA selection. Please make sure that your extraction protocol comprise a DNAse treatment. It is better to perform an additional DNAse treatment if your samples are ongoing rRNA depletion. We have no means to detect DNA contamination in the samples.

RNA quality will be assessed by Bioanalyzer RNA 6000 pico chip (Agilent technologies). Total RNA is considered high quality when the RIN (RNA Integrity Number) is >8. If you performed such analysis, the results can be helpful to us. We will be happy to give you our opinion about it prior to sample submission.

For polyA selection based libraries, RIN should be >8. A 7<RIN<8 might be considered acceptable if your material is difficult to obtain but be aware that the representation of mRNA species might be biased. We do not recommend to start from RIN<7 total RNA.

For rRNA depletion (Ribo-Zero) any RNA can be processed. Degradation may however lead to a less efficient rRNA depletion, resulting in more rRNA reads and thus the need of deeper sequencing than libraries from high quality samples.

Most library preparation kits allow to work with a broad quantity range (ex : 250-1000ng). Under these input quantities recommendations we can not guarantee the quality of the library preparation (it may lead to biased representation of RNA species or high rate of PCR duplicates). Being in the higher range allows to maximize the chance to have higher quality libraries.

RNA-seq with polyA selection (Truseq Illumina Stranded protocol) : 250 ng – 5µg of high quality (RIN>8) total RNA or 50 ng of polyA mRNA. Maximum volume = 50µL

RNA-seq with Ribo-Zero depletion (animal RNA only : Truseq Illumina Stranded protocol) : 200 – 2000 ng of total RNA. Maximum volume = 30µL

RNA-seq with Ribo-Zero depletion (procaryotes/eucaryotes : Illumina Ribo-Zero + Scriptseq protocol) : 200ng – 5 µg of total RNA. Maximum volume = 30µL

RNA-seq from rRNA depleted RNA or polyA enriched mRNA (Illumina Scriptseq protocol) : 5 ng- 100 ng of mRNA. Maximum volume = 20µL

Small RNA libraries :

All RNAs have to be in nuclease-free H2O.
1-5µg of total RNA. Volume max. = 15 µL in water.
Degraded RNA may lead to lower quality libraries

Other type of libraries :

please contact us

par Marion Blin - publié le , mis à jour le