We recently had the opportunity to buy MiSeq, not from Illumina, but from the previous owner. At first, it looked like an interesting offer, but it became a little nightmare. So, I thought I would try to share experiences. Maybe somebody knows somebody who can find someone who can ...?
You may have noticed our June 2017 Statement, where we informed you that, in spite of the near-term end of support for the popular and widely used Applied Biosystems 3130/xl sequencers by the manufacturer at the end of 2018, we at SEQme guarantee their service and application support even after that date.
Our constant search for the best and most appropriate solutions to various experimental designs has led us to the introduction of the GemCode technology into our portfolio of Next-Generation sequencing services. In this post, we are happy to announce its availability to our clients and provide its basic description.
If I had a nickel for every time we answered the “How should I quantify my DNA or RNA for NGS library prep?” question, I wouldn’t need to be writing this article. I’d be on a boat in the Mediterranean. But since I’m not on a boat anywhere, I thought I could perhaps collect several good reasons why using Nanodrop is not really the best way of doing this.
For successful sequencing analysis it is crucial to use optimal template concentration (as described in our guidelines for sample preparation). If you do not have required amount of template, the total volume of a sample can be lowered but the template to primer ratio must be kept. The problem of low template concentration cannot definitely be solved by only increasing the total sample volume...
On March 3, 2015, Life Technologies released a Termination Letter regarding their planned termination of the 3130/xl sequencer support. In this letter, which you may also have received, Life Technologies state that the support of these devices is ensured till "at least up to December 2018"...
Routinely obtained sequence electropherograms typically start with unreliable (unreadable) data just at the beginning of a sequence. Although we should theoretically read the first base after the sequencing primer, there are often errors or truncations just behind the primer ...
Here and there we do receive a request to sequence large templates (i.e. chromosomal DNA, BACs, cosmids) directly. We can do this but similarly as for standard samples where short pcr products or plasmids serve as templates also here the key prerequisite is the template amount.
In a relative quantification experiment, what you are most likely interested in is to compare the expression level of a particular gene among different samples. The most common way of correcting variations in the target nucleic acids input amount among samples is ...
Many of our clients require a regular preventive maintenance. Belonging among them or not, have you ever thought about how you know the maintenance was performed properly?
One of our loyal customers reminded me of a letter he received recently from Life Technologies bringing up a topic dear to my heart – independent service providers! If you’ve been lucky enough to receive this letter too, I would like to take a moment to address concerns you may have.
Have you ever ordered sequencing analysis of a plasmid or PCR product and as a result received the DNA sequence that suddenly stops? One of the possible causes can be that in your template complex secondary structures are being formed, most often hairpins.
Assuming we have good signals and read length is as expected which means we have successfully solved all issues mentioned in previous two parts of this post, we can still be far away from seeing nice data. This third part deals with the problem of having peaks overlapping other peaks. The sequence is not readable.
In the first part we discussed how to troubleshoot no or low signals. Basically empty electropherograms. Very frustrating. Now, I focus on another frequent result – you obtain some signals but the height of the DNA sequencing peaks diminished rapidly. A read length is very short.
It is not the aim of this post to provide a detailed description of all problems you may observe when evaluating your sequencing results. Instead, I focus on most frequent problems and recommend some steps to take for your consideration. Additionally, I am not covering issues related to instruments and sequencing reagents used in our (or any other) sequencing lab because first it is our responsibility to secure a problem-free sample processing on the instrument and second, from the user‘s point of view, it is of low interest because you cannot influence it anyway.
A very frequent scenario when cleaning up your samples (plasmids or PCR products) for DNA sequencing is to load your DNA to silica-based membrane in spin columns. Unfortunately some columns (even those of established manufacturers) may not have a really favorable shape and consequently your DNA gets dirty during purification.
The technology of next-generation sequencing produces huge amounts of data compared to Sanger technology. Its volume naturally depends on the design of the experiment, but primarily on the output capacity of the instrument. In principle, it is always necessary to deal with the transfer of large amounts of data into a form which enables their effective processing to allow a deeper analysis of the sequences obtained which is the very aim of the experiment.