NGS Expert Blog

The third de novo sequenced genome in our series Whose genome has been sequenced? is the “living fossil” Latimera chalumnae.

The most difficult part for this de novo genome sequencing approach was to get enough starting material. The authors even reported that their first approach was to use the Sanger technology, but is simply was not enough DNA available. Therefore they had to wait until the next generation sequencing techniques were stable enough to risk the sequencing (BioTechniques). Here are the sequencing facts of this study (Amemiya et al.):

What was sequenced?

A blood sample from an adult African coelacanth

De novo sequencing strategy:

1. Libraries: shotgun library 61-fold coverage; 3 kb jumping library – 88-fold coverage, 40 kb fosmid library 1-fold coverage
2. Illumina HiSeq 2000 (paired-end module)
3. De novo genome assembly using the software ALLPATHS-LG
4. RNA sequencing

RNA-Seq sequencing strategy:

1. 4 cDNA libraries (1x mRNA-Seq library, 3x strand specific dUTP libraries from brain, gonad/kidney, gut/liver tissue) were sequenced using a HiSeq
2. Data output: mRNA-Seq library ~ 210M paired-end reads;  dUTP libarires ~ 3-4 Gb of sequence/tissue
3. Assembly was performed using Trinity

The genome sequencing helped to understand the possibility of this prehistoric fish to thrive on dry land and the phenotype that is so similar to 300 million year old fossils (BioTechniques).

Read the complete publication here.

Earlier published genomes:

• Goat genome (Capra hircus)
• Chickpea plant (Cicer arietinum)

Domestication of goats happened already thousands of years ago. Nowadays they are also used as models for biomedical research. However, one thing was still missing: a reference genome. Researchers from China could now close this gap by successfully sequencing the genome of a domestic goat.

To reveal the secrets of the goat genome the researchers applied a hybrid approach of Illumina shotgun sequencing and whole genome mapping (WGM) using the Argus system from Opgen. As a result, the number of scaffolds could be reduced from 2,090 to 315. This demonstrates that whole-genome mapping for large genomes can be a replacement for traditional genetic maps for de novo assembly (Dong et. al).

This reference genome can now be used for mapping reads of other goats to identify SNPs and other variants that could play a role for breeding, cashmere fiber prodcution or different goat behaviours (Dong et. al).

If you are interested in more information about optical mapping, read our dedicated blog posts: What is optical mapping? and Creating the perfect genome assembly.

Watch out the presentation of the SITN Boston talking about whole genome sequencing and its impact on personalised medicine.

Further recorded lectures given by graduate students at Harvard and focusing on hot topics in science research and news can be found at https://sitn.hms.harvard.edu/seminar-archive-2012/. Enjoy!

At the end of August, Mr. Hosono, the Japanese minister for the environment, announced, that the ministry aims to perform whole genome sequencing (WGS) of people who live around the disabled “Fukushima Daiichi Nuclear Power Station”. He said that the WGS project will not be able to relieve concerns immediately, but it will make an important provision for the future. According to Mr. Hosono the main target group for WGS will be children.

These genomic analyses face many problems including the aspect of experiments with humans, maintaining confidentiality, discovery of information according to need, and others. This story reminds me once more that NGS technologies start to have social impacts.

In our latest poll we asked you about your knowledge and experiences with whole genome mapping.

As you can see in the pie chart there is no clear answer to that question. But most of you have heard about it and like the technology behind. For all of you who haven’t heard about it I can recommend you our recent blog posts about whole genome mapping using the OpGen technology:

• What is optical mapping?
• Creating the perfect genome assembly

For our next survey we would like to know your opinion about comparing different NGS technologies. I look forward to your answers.

In 2011, University of Münster researchers reported their sequencing of an Escherichia coli strain behind an outbreak in Germany, showing that real-time sequencing was feasible. Then they characterized a K. pneumoniae strain that infected patients in a Dutch hospital. This information was used to develop a multiplex PCR-based assay for the disease.

For the K. pneumoniae outbreak at the US National Institutes of Health’s Clinical Center, Julie Segre and her colleagues sequenced isolates from the 18 infected patients using a 454/Roche machine. From their Newbler assembly and subsequent analysis, they found that isolates from patient 1 and patient 2 differed by two SNPs out of the 6 megabase K. pneumoniae genome, indicating they were infected with the same strain. …

As the cost of sequencing is dropping, major medical centers could soon implement whole genome sequencing to monitor disease exposure of patients. “If the analysis is that you are spending $100,000 to have an organ transplant or go through some very intensive cancer treatment that involves a few nights’ stay in the ICU,” Segre says, “at that point, putting up $500 to sequence an organism that may be affecting another patient in the ICU. … I could imagine in the future that that becomes part of the economic model.”

Read the whole GenomeWeb article at http://bit.ly/Qu3tRX

Six floors for Next Generation Sequencing in the middle of Manhatten – this is going to be exiting. Listen to the interview from Bio-IT World with Nancy Kelly, founding executive director of the New York Genome Center.

Is sequencing your personal genome a curse or a blessing? A recent radio broadcast from NPR news summarises two scientist’s opinions and their practical experiences with genome sequencing  (listen to the radio broadcast below).

World renowned scientist James Watson, from the famous Watson & Crick team that discovered the DNA structure, recently sequenced his own genome. His discovery didn’t earn him the next Nobel prize for science, but he found out that he belongs to the elite few people whose body is more sensitive to ß-blockers. Now James Watson finally realized why it was so difficult for him to balance his blood pressure. It definitely paid off for Watson to sequence his own genome since he could significantly reduce his weekly ß -blocker intake. But despite this “health-changing” experience, he forbid his colleagues to reveal any information about his likelihood to develop Alzheimer. He said, “since you cannot cure it why would you like to know about it?”

The second candidate to share his experience after he personally sequenced his genome is Stanford geneticist Michale Snyder. His genome sequencing revealed that he was at high risk to develop Type 2 diabetes. A few months after his discovery, Synder got the disease that his genome anticipated. Was this a coincidence or fate? For Snyder, knowledge about his genome gave him a head start against the disease.  By completely transforming his diet and participating in various sport activities,  he overcame his Type 2 diabetes.

From my perspective, both examples show that knowledge about our genetic information can be useful in preventing and treating diseases. It boils down to how much experience exist to reliably interpret the data.

BGI announced the acquistion of Complete Genomics this week. According to the press release and the statement from BGI’s CEO Wang Jun, Ph.D.  one of the reasons for the acquistion was that “Complete has developed a proprietary whole genome sequencing technology … (that) fit well with our research and business requirements …”. It is important to note that although the Complete Genomics technology shows a slower performance than the Illumina technology it appears to be more accurate (Georg Church, Advisor of BGI & CG).

But what are the market/industry responses to the new acquisition?

• BGI gets “an immediate infrastructure and service offering that will complement the facilities in China” (Isaac Ro, Goldman Sachs)

• “Now BGI gets their US front… And CG is sparred a slow and painful demise…” (Seqanswers)

I would really like to read an interview in 6 months from employee’s of both parties to learn who is the actual “winner” of this deal? Is it BGI since they now have access to the US market or Complete Genomics whose questionable financial situation gained a significant boost from the acquistion. Will Complete Genomics still be an independent operating company? We will find out.

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.