SAMPLE REQUIREMENTS

Sample amounts

Please find the individual for each library prep sample requirements in the respective tab in the menu on the right.

As a general rule, more DNA/RNA is better, so please provide more than the recommended sample amounts listed below if you can. Using the minimum amount increases the chances of failed preps. We may also be willing to attempt library preparation with amounts lower than the given minima, but this must be agreed with us in advance of submission, and you must nonetheless pay the cost of consumed reagents in the event of prep failure.

Samples with high genomic DNA concentrations (>1 µg/µl) cause problems with our automation due to their viscosity, so are not acceptable. Please dilute your samples to under this limit and re-check their concentration. Do not send DNA or RNA samples, even at high concentrations, in less than 5 µl, as this makes use of automation or multichannel pipettes difficult.

Sample integrity

RNA: Agilent Bioanalyzer RNA Integrity Number (RIN) should be > 7

Genomic DNA: for Nextera DNA flex prep, the sample should have high MW with no degradation. For other preps, fragmented DNA (as from FFPE samples) is acceptable, as the DNA will anyway have to undergo fragmentation by sonication prior to

Sample purity

For DNA and RNA samples the 260/280 ratio should fall in the range 1.8-2.1 and the 260/230 ratio within 1.8-2.4. Different requirements apply for ChIP samples - please see the application-specific tab.

Note that purity measurements (spectrophotometer absorbance) must be documented for each sample in either section 3 of the Submission form (if less than 16 samples) or in The Sample submission table (in Excel format, that you can download from the Forms section on this webpage) (if more than 16 samples).

Sample concentrations

We do not insist that you equalise the concentration of all samples in your submission. However, your samples will be processed faster if you do so, and we encourage this practice. Please see the application-specific tab for required sample amounts and accepted buffers.

Accepted buffers

See the application-specific tab for exact information. Generally, except where specified, do not use buffers containing EDTA, as this will inhibit the enzymatic steps of sample library preparation. If you must concentrate your samples to achieve the recommended minima below, please bear in mind that the final concentration of buffers will affect downstream performance. Do not submit samples in >10mM Tris or other concentrated buffer. We do not accept lyophilized or precipitated samples. Samples delivered in inappropriate buffers will either be returned to the user or subject to additional cleanup costs of 1000 NOK/sample at our discretion.

Accepted tubes/plates and labeling

Each tube/plate must be marked with the user’s name and date, in addition to sample name. When submitting >16 samples, use 96-well PCR plates, not 8-well stripes or 0.5 ml tubes, (filling in columns – see figure below). Lower numbers of samples should be submitted in 1.5-ml Safelock Eppendorf tubes (please don`t tape Parafilm around the tubes). We do not accept precipitated/dried samples.

Correct layout for filling samples in 96-well PCR plates (i.e. filled by columns from left side, not  by rows). Do not leave empty wells or gaps!

Failure to follow these guidelines voids any guarantees on library preparation or run yields!

 

NSC CAPACITY

You may perform sample library preparation in your own lab, or you may submit samples to us for preparation. Subject to our agreement, you may also send trained personnel to perform the preps under our supervision. We operate pipetting robots to maximize throughput, and continually aim to increase the number of samples we can prepare as a service. However, not all protocols are suitable for automation, so we are in some cases obliged to ask that you contribute manpower when the number of samples exceeds our capacity. You should contact us before submission if you have in excess of the following sample numbers:

Sample prep type                    Number of samples / submission

DNA                                        96

RNA                                        96

ChIP / low-input DNA             48

Small RNA                              24

16S                                         940

These limits are subject to revision as we add to our automation capabilities. Higher sample numbers may be handled, but require our prior agreement. Ensure you have downloaded our most recent guidelines to guarantee you are viewing the most up-to-date information from www.sequencing.uio.no.

 

SAMPLE STORAGE

We will store your submitted samples for a period of 3 months following submission. Samples cannot be returned to users except in exceptional circumstances. We store your completed libraries for at least 1 year following project completion to allow further sequencing if requested.

 

PhiX BLENDING

It is standard practice to spike Illumina sequencing runs with 1% PhiX library, which acts as a reference for sequencing performance. The level of PhiX blend must be increased for low-diversity samples, up to 50%, at the expense of yield. If you are concerned that the use of PhiX will cause problems for your downstream data analysis, it is your responsibility to tell us this and request an alternative solution.

Published Mar. 23, 2020 12:45 PM - Last modified Apr. 7, 2020 4:42 PM