Illumina & Aviti Sample and Library Requirements

Please also see the general information on “How To Get Started” and our Sample Submission page.

Library Requirements For Short-Read Sequencing

We are sequencing Illumina sequencing libraries on both, Illumina and AVITI sequencers.  The requirements for both types of sequencers are very similar. The AVITI tolerates longer insert libraries. It also requires higher sequencing library concentrations which are still easy to achieve, especially when pooling several libraries.
The Sequencing libraries should be quantified by a fluorometric method (e.g. Qubit, PicoGreen) or by qPCR.  If a spectrometer (e.g. Nanodrop) is used, we suggest submitting twice the requested amount of sample since this type of measurement is often unreliable. We will quantify all library pools before loading them onto the sequencers.  In some cases the MiSeq sequencers can be loaded with lower library concentrations – please inquire with us.
Libraries should be submitted with Bioanalyzer traces (or equivalent).  We can also run this QC for you, for a fee. The preferred buffer for sequencing libraries is EBT (10mM TRIS, 0,1%Tween20) but EB buffer (10mM TRIS ; pH= 8.0-8.4) is also an option.  The samples need to be submitted in clearly labeled 1.5 microcentrifuge tubes (if not submitting 96 well plates). We highly recommend low-retention tubes. For more information see the Illumina Library Sequencing page. and the Sample Submission page.

The AVITI demultiplexing software is “smart” and will figure out the correct i5 index read orientation on its own.
PCR-Free Libraries – special QC required – The quality of un-amplified libraries is difficult to assess. The adapters of these libraries are partly single-stranded. Thus, they tend to migrate slower than the fully double-stranded, amplified libraries on the Bioanalyzer. In most cases the libraries appear to be 70 to 100 nt longer than they actually are – however the bioanalyzer traces can also be off by far larger margins (e.g. 500 bases). To be sure about the actual library fragment lengths we ask you to to PCR-amplify an aliquot (1 ul) of the libraries with 8 PCR cycles and run both the PCR-free and the amplified sample on the Bioanalyzer. If multiple PCR-free libraries will be pooled, you might consider quantifying the individual libraries by qPCR before pooling.
Custom sequencing primers (very few assays require these) need to be submitted at a concentration of 100 uM and a volume of 20 ul each together with the libraries. Please make sure that the sequencing primer design fits the chosen Illumina platform. Miseq and Hiseq platforms use different annealing temperatures.
Demultiplexing is included with our sequencing services, however please ensure your sample IDs and barcodes are unique; we are now implementing additional labor charges for re-demultiplexing because of incorrect submission info, duplicate sample names, wrong barcodes given, reverse complements given in error, etc.

DNA/RNA Sample Requirements for Illumina Library Preps

The input DNA and RNA quantities specified below apply if the samples are quantified by a fluorometric method (e.g. Qubit, PicoGreen, RiboGreen). Fluorometry provides big advantages in precision and specificity (e.g. DNA dyes will not bind to/measure RNA). If a spectrophotometer (e.g. Nanodrop) is used, we suggest submitting twice the requested amount of sample since this type of measurement is often unreliable. In any case, sample amounts higher than the minimum requirements will improve the library complexity and thus the quality of the data. Spectrophotometer/Nanodrop readings are required to assess the purity of samples. NEVER use heparin as an anticoagulant for blood samples destined for DNA or RNA sequencing. EDTA (preferred) or citrate anticoagulants should be used.  DNA samples for Illumina sequencing should be isolated with spin-column protocols (e.g. DNeasy; multiple vendors offer similar kits).  Such kits are also available in plate format for high-throughput processing.

The table below provides an overview over the sample requirements.  For additional details please see the Illumina library prep page and the PacBio sequencing page.

Please note that the table indicates both Recommended Sample Quantities as well as the Lowest Quantities.  The Illumina sequencing library prep protocols become less efficient with low sample quantities, but they do not fail at a certain threshold.  Further all Illumina sequencing libraries can be PCR-amplified.  For some applications (e.g. small genomes) even lower input amounts might still generate usable data. Please contact us to discuss such situations. However, to avoid unnecessary PCR-cycles and potentially low-complexity sequencing libraries please follow the recommended sample specs for standard applications.
For more information see the Illumina Library Construction page.   Samples for the high-throughput library preparations (at HT rates) need to be normalized to +- 20% of the average sample concentration.

RNA samples need to be DNA-free and should be dissolved in molecular biology grade water (RNAse-free; not DEPC treated).
DNA samples need to be RNA-free  and dissolved in EB buffer or TLE buffer (please see below). Molecular biology grade water is also OK  for Illumina samples.

Please note that an additional column cleanup is mandatory for RNA samples isolated from blood sample PAXgene or Tempus tubes (for blood sample preservation)  or with the accompanying PAXgene and Tempus RNA isolation kits.

We require the submission of isolated total RNA or DNA samples. We do not perform RNA or DNA isolations for Illumina sequencing in our lab, but the PCR-lab in the neighboring building does; please contact them.

PacBio Sample Requirements for Genomic Library Preps

For more information see the PacBio Library Prep & Sequencing page.

PacBio Isoseq: We recommend submitting 1 ug (or more) per total RNA  sample (DNA-free) at a concentration of 50 ng/ul or higher in molecular biology grade water. The nanodrop  260/230nm ratio should be >1.5 and the 260/280nm ratio 1.8-2.1.  RIN-score 8 or higher. For plants 7 or higher.

Sample QC Requirements

For more information see the Sample & Library QC page.
Please note that samples for the Bioanalyzer have to be detergent-free (PCR-buffers contain detergents and other enzyme buffers may also; such samples have to be purified before submission).

See this FAQ for information on RNA-QC (fragment analysis) on the LabChip GX.

Batch RNA-QC on the LabChip GX (most Wednesdays; cost per sample $8 for UC labs.

Requirements for Batch RNA-QC:

  • Provide sample names with an implicit order. The traces will identify the well position not sample names.
  • We will not adjust sample concentration or volumes.  It is your responsibility to meet the sample requirements.
  • submit total RNA samples in strip tubes (we can provide some if necessary) and a filled out QC submission form.
  • each RNA sample needs to have a volume 2 ul to 6 ul and contain 30 ng to 250 ng total RNA (this is the amount not the concentration).

Buffer Compositions

Common buffers for DNA and RNA samples are:
EB-Buffer: 10mM TRIS (pH= 8.0-8.4) – e.g. Qiagen EB Buffer
EBT-Buffer: 10mM TRIS, 0,1%Tween20 (pH=8.0-8.4)
TE-Buffer: 10mM TRIS, 1 mM EDTA (pH=8.0-8.4)
TLE-Buffer: 10mM TRIS, 0.1 mM EDTA (pH=8.0-8.4)