Library Requirements For Illumina Sequencing
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 and HiSeq 2500 sequencers can be loaded with lower library concentrations – please inquire with us.
Libraries should be submitted with Bioanalyzer traces (or equivalent). We can run the Bioanalyzer 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). Best use low-retention tubes. For more information see the Illumina Library Sequencing page. and the Sample Submission page.
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.
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 islotated 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.
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 purified before submission).
Batch RNA-QC on the LabChip GX (most Wednesdays; cost per sample $5 (with a minimum of $10) for UCD 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).
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)