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New horizons for Next-Generation Sequencing with GemCode technology (10x Genomics)

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.

Several good reasons why Qubit is better than Nanodrop when quantifying your samples for NGS library prep

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.

Low template concentration – What can you do?

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...

Our statement regarding the Applied Biosystems 3130/xl termination letter by Life Technologies

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"...

Higher-quality sequence data at the beginning of electropherogram - Modified sequencing primers

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 ...

Applications of next-generation sequencing

The power of high–throughput DNA sequencing technologies is being harnessed by researchers addressing a very wide range of scientific questions. Options offered these days by state-of-the-art next-generation sequencing platforms stand behind an unprecedented progress in many areas from the analysis of genomes through RNAseq world to how proteins interact with nucleic acids.

How do I sequence large templates directly?

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.

How do you work with .ab1 files?

Results of Sanger sequencing are provided in three types of file formats and not every user of our services is familiar with efficient usage of these data files. Here are brief instructions how to use them properly.

Is there a need for the reference gene(s) to be measured in the same plate (run) as the gene of interest?

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 ...

Do you own Applied Biosystems 7900HT? Lucky you!

Have you ever seen the Applied Biosystems 7900HT Real-Time PCR system? Without really knowing what you are looking at, you probably didn’t like it. In my opinion this is the ugliest qPCR instrument ever, grey, huge, heavy and noisy.

Preventive maintenace of your sequencer – what do you expect?

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?

A letter signed by life (ooops, my mistake - Life)

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.

HairpinSeq – a sequencing protocol for difficult templates

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.

10 REASONS to Sequence at SEQme

Are you wondering why you should send samples to us? Please read our 10 good reasons to answer this question!

A Few Thoughts on Troubleshooting of DNA Sequencing - Part III

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.

A Few Thoughts on Troubleshooting of DNA Sequencing - Part II

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.

A Few Thoughts on Troubleshooting of DNA Sequencing - Part I

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.

Are you contaminating your DNA on cleanup columns?

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.

A few thoughts on data analysis

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.

Short Glossary of NGS terms

Brief explanation of commonly used NGS terms

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