NGS Expert Blog

Last week The Smithsonian Institution’s National Museum of Natural History at Washington DC announced a new exhibit to celebrate the 10th anniversary of the completion of the human genome. The project is a collaboration between the museum and the National Human Genome Research Institute, with major funding coming from the Life Technologies Foundation. It will open in 2013 to the 7 million annual visitors of the museum.

“The goal of the exhibition is not just to celebrate but to look ahead and acknowledge that we are in the early stages of a very exciting genomic era, that we have learned a remarkable amount about how the genome works and how it contributes to health and disease, and that the pace of research is only accelerating and becoming increasingly relevant to people,” said NHGRI Director Eric Green.
Also announced last week was a new grant program by the NHGRI to study newborn genome sequencing. It will provide $25 million to study how whole genome or whole exome sequencing will benefit newborn care as well as its social implications.
“Genome”, “genomics” etc used to be terms understood by few outside of biology and bioinformatics. This is changing rapidly. It is exciting time ahead of us to witness the genomics revolution.

Some time ago I was introducing a new approach combining restriction site associated DNA marker genotyping (RAD) with next generation sequencing technology. Originally this method was developed for microarray platforms. However, the combination of RAD and NGS (Illumina) – resulting in RAD sequencing (RAD-Seq) – enabled the massivly parallel and multiplexed sample sequencing. RAD-Seq is becoming more and more powerful and has the potential to revolutionize agrigenomics, because one can discover and screen thousands of SNP’s and genotype large populations in a high throughput manner at the same time. The scope of the following section is to give a short technical overview how this can be accomplished:

Genomic DNA of each sample is digested in parallel with a certain restriction enzyme and a specific P1 adapter is ligated to the restriction fragments. Thereby each sample will be equipped with an individual P1-adapter containing a sample-specific molecular identifier (Barcode) and Illumina adapter sequences (forward amplification primer site and Illumina sequencing primer site, respectively). If multiplexing is desired, the adapter-ligated fragments of a number of samples can now be pooled. The level of multiplexing depends on the number of differed P1-adapters which have been used before. In a further step the RAD pool will be sheared, size-selected and ligated with a second adapter (P2). The P2 adapter comprises a divergent “Y” adapter containing the reverse amplification primer sites. However, the P2 adapter is special such that fragments lacking the P1 adapter cannot be amplified. This guarantees, that only fragments containing a P1 and a P2 adapter will be selectively and robustly enriched during amplification step following next. The overall length of RAD-tags which can be further analysed mainly depend on the size selection step and sequencing run mode (single vs. paired end), respectively.

My last week’s blog article was about expression profiling with mRNA-Seq libraries and about the required sequencing depth of this protocol. But there are other possibilities for expression profiling, and today I especially want to highlight the 3’-fragment library protocol.

The big advantage of this protocol is that it provides a much higher resolution than mRNA-Seq does. The reason is that within mRNA-Seq the average transcript is represented by approx. 10-25 reads that cover the whole transcript, while with the 3’-fragment protocol only one read is generated per transcript. The derived reads from a 3’-end library map to the 3’-end of the transcripts and expression differences are easily collected by just counting the reads that map to a specific reference transcript.

The 10-25-fold higher resolution comes along with considerably reduced projects costs as 10-25-fold less sequencing is required to obtain a similar depth of the analysis. Or in other words: When analyzing the same number of samples per channel the 10-25 fold higher resolution allows the scientist to even look at very low expressed genes with reliable statistical evidence.

Of course the mRNA-Seq protocol is needed in case other analysis shall follow, like the study of alternative transcripts, or fusion genes. But this is anyway a completely different story as these applications need an even higher sequencing depth than expression profiling with mRNA-Seq does require.

As a conclusion I think it is definitely worth to evaluate this protocol when having in mind an expression profiling experiment. And we would be delighted if you share your thoughts on this with us and the other blog readers.

The majority of scientists performing expression studies use the mRNA-Seq protocol (random-primed cDNA synthesis after fragmentation of PolyA-purified transcripts) and sequence the fragments with Illumina technology. By planning the experiment the question of the sequencing depth immediately arises. And for all of you being interested in an answer I want to share with you the recommendations on sequencing depth from experts in the field of transcriptome sequencing published in genomeweb.

You can see that the recommendations vary between 10 million single end reads and up to hundreds of millions reads depending on the exact need. And it is really tough for the experts to give a concrete number. Please keep in mind that about 80-85% of the transcripts in a typical transcriptome are representing only a few highly expressed transcripts whereas the majority of transcripts is present in a few copies only. For just straight gene expression analysis the interviewed scientists usually use around 20 – 30 million reads per sample. But when your aim is to look at really low expressed genes, like some transcription factors, you definitely have to apply a higher sequencing depth. And the very same is true when transcript isoforms or fusion genes shall be analyzed. For this applications the required sequencing depth can be as much as a full channel per sample.

So, enjoy reading their comments!

• Would you like to detect variances down to a frequency of 0.1% in your PCR sample?

• Are you interested in a short turn around time?

• You need primers for your amplicons?

Amplicon sequencing is still under discussion. Which technology is most suitable? In one of our latest blog posts we discussed this issue as well. For this years spring special we therefore decided to create a new NGS Favourite – a one stop solution for Amplicon sequencing. By sequencing your amplicons on the GS Junior we will be able to deliver the data in a short turnaround time while you will still profit from the long reads the FLX chemistry provides. Furthermore you will get comprehensive bioinformatic data that will be able to answer already questions like: how many clusters were obtained or what is the homology of each read compared to the representative read. This data will help you for example to analyse metagenomes in your environmental samples like soil, water or gut.

Additionally we as a service provider for oligonucleotide synthesis, gene synthesis, custom DNA sequencing and NGS are able to offer you primers for free for your amplicon sequencing approach if you order our Spring Special.

Contact us if you are interested in a spring special quote or read more on our website. This offer is valid until 30.06.2012.