Illumina TruSeq DNA Nano
Library preparation from limited input DNA, used in whole genome sequencing and metagenomics etc.
Illumina TruSeq DNA Nano library preparation is a library preparation method for samples with limited DNA amount.
This method is recommended when:
- You have limited amount of starting material (< 1000 ng).
- You want to adjust the insert size of the library. Different insert sizes are currently available: 350 bp (standard) and 550 bp.
- Sample type: high-quality DNA
- Amount of sample: minimum 750 ng
- Volume: 25-50 µL
- Amount of material needed for preparing one library: 100 ng (350 bp) or 200 ng (550 bp)
How to evaluate the sample quality
We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples.
Checking the concentration
Please use fluorometric measurements (e.g. Qubit, Quant-it), not absorbance measurements (Nanodrop, spectrophotometer).
Checking the quality and purity
The DNA should be in water or EB buffer, of high quality, and ideally have an OD260/280 of 1.8-2.
If your samples are below the required thresholds, please get in touch.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the samples meet our requirements.
If the samples fail this quality control step, we will contact you to discuss possible options. Should you choose to proceed with samples not fulfilling the criteria it will be at your own risk. NGI will only make one attempt at library preparation and if the library preparation does not work you will be charged for the library preparation regardless.
If the samples pass reception control, the samples will be queued for library preparation.
The protocol for DNA library preparation is automated. The DNA is sheared on a Covaris sonicator followed by a size selection using AMPure beads. Illumina adapters with sample-specific barcode sequences are ligated.
Library QC and sequencing
We evaluate the concentration of the libraries using qPCR and look at the library fragment size distribution of the library molecules using TapeStation or FragmentAnalyzer. Once the libraries have passed this QC step, they are normalised, pooled, and queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.
In general, we have been successfully generating libraries that provide enough good quality reads for sequencing of genomics DNA. However, the sequencing results from each library prep will depend on the characteristics of the sample:
- The yield of the library preparation could be insufficient when the input is below our input criteria or if the DNA is degraded.
- When the library concentration is too low, generating an even pooling is more difficult, so you can expect uneven reads yields.