How tos and FAQs

Guidelines for Ready-to-Run libraries (Illumina)

When analyzing your samples by Next-Generation sequencing, we prefer you send DNA or RNA. For various reasons we want to prepare sequencing libraries in our lab. But obviously, another option is you can perform this part of the workflow yourself and you will be sending sequencing libraries to us (Illumina technology only!). We call them Ready-to-Run libraries and below are our requirements for this scenario.

  • Customer-prepared libraries must be provided as a final pool (we do not perform pooling of libraries which we do not prepare). 
  • Individual libraries (sub-libraries) must bear Standard Illumina Adaptors*.
  • In case of custom adaptors requiring custom sequencing primers on our end, our tech support must always be contacted first.
  • Information about sequence diversity of inserts and indexes must be provided.
  • Concerning index choice please follow Illumina Index Adapters Pooling Guide*.

All this information must be entered when filling in the online order either directly into the prepared forms or by uploading a file containing these additional data. You will be prompted to upload it when ordering. An order form for this type of analysis is available at this link.

Ready-to-run libraries cannot be processed by using the ShareSeq protocol.

* newest editions of both guides can be downloaded from Illumina website.


Remarks:

It is highly recommended that libraries are of rather narrow size distribution. This is especially true when the sequencing library is a pool of sub-libraries. If you do not meet this requirement, the inevitable consequence is the yield bias because Illumina sequencers use diffusion based loading method and as a result positions at the sequencing flowcell are occupied by shorter fragments slightly more frequently than by longer fragments.

This should not cause very big trouble for a shot-gun library or uniqe size amplicon library occupying whole chip or lane. But once you pool together libraries or amplicons of different sizes, you can expect higher sequencing yield for shorter libraries/products than for those that are longer. The picture below shows size distribution of prepared and sequenced fragments. Fragment size distributions are shown for two libraries, the red lines represent the size distributions reported by Agilent 2100 Bioanalyzer, the light blue areas represent inferred size distributions of the sequenced fragments.

(Picture adapted from Hara Y, Tatsumi K, Yoshida M, et al.; Optimizing and benchmarking de novo transcriptome sequencing: From library preparation to assembly evaluation; BMC Genomics 2015, 16: 977).

When in doubt, please contact us!

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