Illumina sequencing

NGI can sequence user-prepared libraries on all Illumina instruments. User-prepared libraries orders can only be whole-lane orders. Libraries with custom setups and or custom primers can only be whole runs or whole flow cells.

Sample requirements

If you have prepared your own Illumina-compatible sequencing libraries, then we require at least 20 μl of 10 nM library pool for a MiSeq/NextSeq run, and 20-70 μl for a NovaSeq run. The range of fragment sizes within the library pool should be >read length requested and <800 bp, no traces of primer dimers etc remains.

If you require sample de-multiplexing please provide us with the index sequences used.

Custom primers

It is possible to use custom sequencing primers or a combination of custom primers and Illumina primers provided in the reagent cartridge. Please supply min. 15 μl of custom primers at a stock concentration of 100 μM. Please contact us if you have questions regarding custom primers.

How to evaluate the sample quality

We will do reception control upon samples arrival, however we still require our users to do their own QC steps before sending samples. We require Bioanalyzer (or similar) traces of the submitted library pool. The scale on the x-axis must be in basepairs. We will evaluate the library concentration by qPCR.

If you are not able to provide a Bioanalyzer trace, or your samples are below the required thresholds, please get in touch.

What we do with your samples

When receiving final libraries, we first determine the concentration by qPCR measurement plus the fragment size information. Then we denature and dilute the library and add PhIX spike-in. We normally add 1% PhIX to all Illumina runs, but for libraries with low base-complexity (such as amplicons) a higher PhiX spike-is is recommended (usually ca 10%).

We perform a QC check on the sequencing run. If the run failed the sequencing run QC, we will re-sequence the samples that passed our reception control. If the sequencing run itself is failed (stopped during the run etc), we will start a new sequencing run no matter the sample failed or passed our reception control.

Expected results

Please see respective sequencing platform under “relevant technologies” in the sidebar to the right.

Bioinformatics

We will follow our bioinformatics analysis guideline to do the sequencing run QC and the demultiplexing.

Applications
Relevant Technologies
Bioinformatics Pipelines
Method Status

Service

We are routinely running this method. Please visit the Order Portal to place an order.

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