NGS Expert Blog

Several companies have announced in 2011 to launch Trisomy 21 tests based on Next Generation Sequencing as described in a blog post from May. I have a short update for you on what has happened in the diagnostics market since May.

In fact only several months after these announcements the first test has been brought to market by Sequenom. The test has been launched in October 2011 and company officials have disclosed that out-of-pocket costs for the test are $235. The turn-around-time for the tests is said to be 8-10 days after sample submission. In a supporting large-scale and blinded clinical validation study the company and collaborators have shown a detection rate of 98.6% and a false positive rate of 0.2% while simulating throughput and turnaround times of clinical practice.

The company Verinata is not far behind. The company is currently conducting a blinded pivotal study to further validate its first prenatal tests to identify multiple fetal chromosomal abnormalities that the company is expecting to launch late this year or early next year.

It remains exciting to wait and see which company finally is able to offer a convenient and affordable test and how the acceptance of the test on the market will look like.

What is RAD-Seq?

Restriction site associated DNA sequencing (RAD-Seq) using the Illumina technology was initially published in PLoS One and in PLoS Genetics. Along with other studies, these studies demonstrated that this smart technique can be very useful for the identification and further analysis of a high number of genetic markers distributed over the genome. This is especially advantageous if working with non-model organisms where no reference sequence is available. Briefly, RAD-Seq results in a reduced representation of the respective genome, because only fragments near a specific type of restriction site are sequenced to deep coverage. Such fragments are called ‘RAD tags’ and serve in subsequent analysis steps as a reference for the design of genetic markers. The overall number of possible RAD tags within an organism strongly depends on the restriction enzyme of choice and the genome of interest.

The poll did run since September 14th. 36 ngs-expert.com readers did submit their votes.

• 22,5% prefer buying an instrument for their NGS applications.

• 25% think that outsourcing depends strongly on the project itself.

• 50% share our opinion that outsourcing is the preferred method. As already posted in September it seems that many instruments are used far below their actual capacity. Since expertise in next generation sequencing, sample preparation and handling improves by regular working with the instruments, the real advantage of having the sequencer in the lab is limited. Based on this it might be more efficient to invest the research funds in a more effective way.

Many large scale exome sequencing projects are funded and underway to analyze rare Mendelian diseases. This technology is often the choice as it is more affordable than whole genome sequencing (WGS) and therefore allows analyzing more patients. In addition it has the advantage that resulting data volumes are much smaller and therefore easier to handle.

But – when looking only on those regions targeted by the exome technology – are the results of an exome sequencing experiment really comparable to a WGS experiment?
 
The study from Clark et al., 2011 focused on this question and found that neither of the technologies managed to cover all sequencing variants. When applying 50 million reads for exome sequencing and 35-fold coverage for WGS, the study came to the following results.

- WGS detected between 660 and 4600 SNPs that were not called from the exome sequencing data and
- Exome Sequencing detected between 2600 and 3200 SNPs that were not called from the WGS data.

What can we conclude from this? First, WGS can not and will not replace exome sequencing as due to genome characteristics there will always be regions that are not covered sufficiently for SNP calling. As oligonucleotide designs of available exomes are balanced regarding regions with low coverage, exome sequencing shows higher sensitivity towards these regions. Second, WGS has its value in detecting variants in regions that are not covered by exome enrichment technologies. These are regions where enrichment fails as well as regions that are not present on the current exome designs.

So for covering really all variants it might be worth thinking about doing both experiments in parallel. Both technologies complement each other.

As a NGS Winter Special we do offer you a selection of our well-proven portfolio for transcriptome sequencing. De novo sequencing of eukaryotic transcriptomes will provide you a deep insight into the transcriptome without the need for a reference sequence. With the expression profiling package you will gain high resolution information about expression levels of your bacterial genes.

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Sequencing in 1 channel with 2x 100 bp paired end module

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Expression profiling of 12 bacterial transcriptomes on Illumina HiSeq 2000

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All Services include library generation, data analysis & data delivery. The Winter Special is valid until 31.12.2011

Read more about our NGS Winter Special >