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FAQ

You are welcome to contact use via email with specific questions. However, we recommend that you first take a look below at a compilation of questions that have been asked and answered before. In addition, you may find it useful to read our short introduction to HTS and the description of our sequencing services .


General questions (sequencing platform independent)

What is paired-end/mate pair sequencing?

A pair of reads coming from each end of the same contiguous DNA molecule are called "paired end" reads, and the distance between the two ends is user-definable within the range of 100 - 700 bp. The term "mate pair" describes pairs separated by a greater distance (typically 2 or 5 kb). Originally spaced by 2 -5 kb, these template molecules have been circularised, juxtaposing the ends, allowing sequencing of the ends of the original molecule in a small construct. Both paired end and mate pair libraries thus produce similar data (paired reads originating from the same template molecule and separated by a known distance) but their different construction method and insert length gives them their differing names.

Do you provide cDNA sequencing?

Yes, cDNA sequencing is available  - these submissions are essentially treated as DNA sequencing projects. In many cases it is best to simply submit RNA to us for prep (by poly-T bead selection of polyadenylated RNA, or total RNA sequencing) rather than preparing cDNA yourself.

What documentation should be submitted with my sample(s)?

A sample submission form relevant to the sequencing technology that you are using should be filled out and submitted as soon as possible before delivery/submission of your samples.


Illumina Sequencing

What are your prices for Illumina Sequencing?

Pricing is dependent on number of samples, indexing, number of lanes, length of sequence required and the NOK:USD exchange rate, so there is no standard price list. Please contact us for a price estimate for your particular project. Estimated prices and actual prices may vary (increasing or decreasing) depending on the exchange rate at the time the reagents for your project were purchased. We can supply quotes, typically valid for 30 days.

Who does the sample preparation?

We are flexible about this. We can do the prep, the user can do the prep, or the user can visit our lab to do the prep.

When will I get my data?

Delivery times vary depending on which type of library prep you request, and our queue. Typical delivery times are in the range of 5 - 8 weeks for projects run on the HiSeq, and 1-5 weeks for those on the MiSeq. We do not have the capacity to provide updates on the progress of your sample in our system, so please do not ask for them. We are an academic facility, not a company, and prefer to use our time sequencing than answering requests - and you will get your data sooner too -  so please be patient!

How long does the sample preparation take?

This depends on the protocol, but from ranges from 1-5 days. In practice, however, samples must wait in a queue for prep.

How many samples can I submit?

If you would like us to perform library preparation, we have maximum limits above which we ask you to contribute to the workload of sample library preparation. These limits are sample type dependent, and the most up-to-date figures can be found in our submission guidelines http://www.sequencing.uio.no/forms/guidelines-submission-form-(illumina).pdf. We operate pipetting robots to automate sample library preparation, and raise our limits as and when we can. If you require assistance to prepare a greater number  of samples than we can offer, you are welcome to contact us for advice on post@sequencing.uio.no

Can prepared samples be stored and for how long?

Completed sample preps (libraries) generally provide enough DNA for several flow cells, and can be stored for at least one year at -20 oC. However, PCR-free libraries, which contain single-stranded DNA, have a shorter life that has not yet been fully characterised.

Can I have different samples on the same flow cell?

Different samples can be sequenced on different lanes of the same flow cell - but all will be sequenced for the same number of cycles.  Samples prepared with different protocols (mRNAseq, smallRNA, ChIP, genomic DNA, etc.) can be run together on a single flow cell. We also have the possibility to multiplex samples (a.k.a. indexing / barcoding), enabling up to 96 samples to be run per lane (depending on sample type). Depending on library size, it is sometimes also possible to combine different sample types within a single lane (e.g. mRNA and gDNA libraries can often be combined, but the different sizes of miRNA and gDNA libraries make these largely incompatible).

How long does a run take?

The length of run depends on the number of cycles of sequencing required but varies from 1 days (50 bp single end) to 3 days (150bp paired end) on the HiSeq 3/4000 or X systems. Runs on the MiSeq and NextSeq are shorter - maximum 65 hours for 300 bp paired end. However, the submission queue, sample preparation and post-run analysis mean that the total time required to perform sequencing is considerably longer. The greatest effect on queue time is due to the requirement to fill full flowcells (8 lanes) on the HiSeq series instruments, which often means that your sample is dependent upon the arrival and completion of other users' samples before a run can commence. MiSeq and NextSeq runs, in contrast, consist of  only a single lane, so run as soon as a machine is available.

What size are the inserts in a PE run?

Insert sizes are user-defined in the range 200-700 bp. It is also possible to produce long-insert libraries (also known as “Mate Pair” libraries) so that fragments 2-5 kb can be end-sequenced (important in many assembly projects). Standard insert sizes are 350 bp or 550 bp - depending on capacity, we may ask users to prepare libraries themselves if they desire non-standard insert sizing.

How much coverage do I need?

This depends on the project - for example, the requirements will be very different for resequencing/mutation detection and ChIP. Users should discuss this with the platform.

How do I get the output data?

Data will be made available for download by ftp. We can also provide data on portable hard drives, which is our standard practice for sensitive (human) data. 

How do I analyse the output data?

There are many tools available to analyse sequence data but as a first step, users should discuss analysis requirements with the platform.

Which analyses do you perform as a service?

We aim to expand our analysis offerings. However, at the moment the only service available (free of charge) is alignment of DNA reads to a reference genome of your specification. This is not performed as a default, and users must request this extra service. We may be able to assist you with additional analyses, but for the time being these must be approached on a case-by-case basis and will generally require an agreement of collaboration with one of our bioinformaticians.

 

How many samples can be indexed (aka bar-coding, multiplexing, or tagging)?

For gDNA and mRNA libraries up to 96 libraries can be indexed and run in the same lane. Up to 384 indexes are possible using the Nextera tagmentation reagents. For small RNA studies, up to 48 samples can be indexed. Higher numbers can also be achieved using reagents from 3rd party suppliers such as NuGen and BioO Scientific.

Further information on analysing sequence data.

Published Mar. 13, 2012 6:01 PM - Last modified Jan. 20, 2017 12:07 PM