Library Requirements For Illumina Sequencing
Sequencing libraries should be quantified by a fluorometric method (e.g. Qubit, PicoGreen) or by qPCR. 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. 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 expected yield metrics apply to standard balanced libraries like RNA-seq or genomic libraries; i.E. high complexity libraries with average GC contents and of the recommended library molecule lengths. For these libraries we do promise that the yields will exceed the Illumina specifications; please see the FAQ.
The standard requirements for library submission are at least 15 ul volume at a concentration of 5 nM (e.g., 1.6 ng/ul given a 500 bp library). More volume and/or higher concentration is welcome. If required, 20 ul of custom sequencing primers need to be submitted together with the libraries at a concentration of 100 uM each. Depending on platform, we can work with less library (down to 1 nM for MiSeq and HiSeq2500), but the quantification becomes less reproducible, the library becomes less stable, and relatively larger amounts of library DNA stick to the sides of the storage tube. Lower sequencing yield is the likely outcome for library concentrations 1 nM or less, and we cannot guarantee the data quantity or quality for such libraries. The best buffer to store and submit libraries is 10 mM Tris/0.01% Tween-20 ph=8.0 or 8.4, but EB buffer is also acceptable. Please use 1.5 ml low-bind tubes (e.g. Eppdendorf LoBind). If you do not provide a Bioanalyzer trace (or equivalent) of your library, we will run the Bioanalyzer for a fee. Please note, the DNA insert size(s) should not exceed 700 bp and most Illumina adapters add about 120 bases to the fragment length as observed on the Bioanalyzer. When submitting your libraries for sequencing, please use our Illumina Sequencing Submission Form (hard copy with samples, and email to firstname.lastname@example.org), provide the Bioanalyzer profile, library prep methods, and index sequences used. We will measure the quantity of your libraries using real-time PCR (included in the sequencing price).
Libraries for HiSeq 4000 and NovaSeq sequencing – The latest generation of sequencers has more stringent library requirements and requires higher library concentrations. For the HiSeq 4000 the average library fragment length should ideally be 420 bp and the “tail” of longer fragments should not exceed 670 bp (with adapters). For the NovaSeq, average fragment length should ideally be between 420 bp and 650 bp and the “tail” of longer fragments should not exceed 1 kb. The longer library molecules will result in lower quality reads for this fraction of the samples and will impact rad numbers reducing the yields. The new clustering chemistry is more sensitive to adapter dimers: a 5% adapter-dimer contamination can result in 60% of the reads coming from these dimers. Thus it is very important that there is no indication of an adapter-dimer peak (around 120 bp) on the Bioanalyzer trace. Our preferred requirements for library submission are for at least 15 ul volume of 5 nM concentration per lane (e.g., 1.6 ng/ul given a 470 bp library). More volume and/or higher concentration is welcome. Lower sequencing yield is the likely outcome for library concentrations 2 nM or less, and we cannot guarantee the data quantity or quality for such libraries.
We highly recommend using uniquely-dual-indexed (UDI) adapters for both the Hiseq 4000 and the NovSeq. Hereby matching i5 and i7 indices per library must be avoided. The UDI adapters allow the efficient elimination of all potential index hopping artifacts.
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.
Library Pooling Service – We offer the pooling of sequencing libraries for a small fee. Prerequisites for the pooling of customer libraries are:
- all libraries were generated using the same protocol and are PCR amplified
- their fragment size distribution is similar (and within the Illumina specs) as demonstrated by Bioanalyzer traces
- uniquely indexed adapters
- all libraries need to have DNA concentrations in about the same range
Library pooling requires precise pipetting of very small volumes and we can’t work magic with wildly variable samples. PCR-free libraries are best quantified by qPCR. Other libraries can be quantified by fluorometry (e.g. Qubit). For sequencing libraries generated by the core labs, the pooling is included in the service. Please also see our FAQs.
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.
Libraries: Quality Control (QC)
Library quality is the single most important determinant of the success of your sequencing run, both in terms of the number of reads generated (quantification) and the validity of the sequence obtained (content). We carry out two QC measures on all libraries sequenced in our Core – examination on the Agilent Bioanalyzer, and quantification using the Kapa Biosystems Illumina Library Quantification Kit. The “perfect” library Bioanalyzer trace, pictured here, shows a single peak of the expected molecular weight. Common additional forms include primer dimers (at around 80-85 bases), adapter dimers (around 120 bases), and broader bands of higher MW than the expected peak. Primer dimers are not a problem unless they completely dominate the reaction. Adapter dimers can be a problem because they will sequence much more efficiently than the longer library fragments. As a result, whatever the proportion of adapter dimers in your library will be seen as an even higher percentage of reads in your final data files. Adapter dimers can be minimized by adjusting the adapter:insert ratio during library construction and exercising care in gel extraction, bead purification or other size selection steps. The larger MW, typically more hump-shaped forms that are visualized on the Bioanalyzer are probably a result of excess amplification during the final PCR step. While some amount of these are tolerable, if they are too prominent then the library should be re-amplified from the gel extracted material.
The electropherogram of a sequencing library should show a distinct peak. The tighter the library fragment size distribution is, the more accurately the library can be loaded onto the flowcells.
Library QC for PCR-free Sequencing Libraries PCR-free libraries pose special problems since they can show several types of misleading artifacts on Bioanalyzer runs. Thus, we require a Bioanalyzer trace of the library to be sequenced, as well as a trace of an amplified aliquot of the same library. Please see this section of our FAQs for the details.
Custom Sequencing Primers Please note that these are used only for a small minority of sequencing projects. Custom sequencing primers 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.
All customer submitted libraries should be accompanied by Bioanalyzer (or similar) traces. If no traces are submitted we will carry out the Bioanalyzer analysis for a fee. Please visit the Sample Submission & Scheduling page to download submission forms and for detailed instructions.
Once your library, or library raw material, is ready, you should deliver it as soon as possible to get the next available slot in the queue. Runs occur as we fill up the two (rapid mode) or eight lanes on a HiSeq flow cell, and the timing on this can vary depending on service type and Core activity. For MiSeq runs the turnaround time is typically five to eight days, while you should allow three to five weeks for HiSeq sequencing; in both cases allow an extra one to two weeks for library prep. The HiSeq sequencing scheduling calendar is now available on our website.
Sample/Library Storage Policy
Please let us know if you would like to pick up your samples/libraries after they have been sequenced and we will be happy to accommodate you; otherwise, due to space limitations, they will be stored for only six months after sequencing runs have been completed.