Clinical Genomics Using NGS Approach

Video by Cambridge Healthtech about the impact of next generation sequencing on clinical genomics.

Nazneen Aziz of the College of American Pathologists and Konrad J. Karczewski of Stanford University are talking about analytical and bioinformatics standards, personal analysis, challenges and interpretation services.


Whose genome has been sequenced? Nasuia deltocephalinicola

de-novo-sequencingThe human genome comprises more than 3 billion base pairs and builts up more than 20,000 protein coding genes. For genomes like this high-throughput sequencers, like the HiSeq 2000 are a revelation. In this article we talk about the smallest genome sequenced so far – here sequencing with the MiSeq is more than sufficient. The main role of this small symbiont (Nasuia deltocephalinicola) together with the symbiont Sulcia muelleri is to provide 10 essential amino acids to the pest insect Macrosteles quadrillineatus (Bennett et al).

What was sequenced?

10 phloem-feeding pest insects (Macrosteles quadrillineatus) including the obligate symbionts Nasuia deltocephalinicola & Sulcia muelleri.

Sequencing strategy: Whole genome sequencing

  1. Libraries & Sequencing: 2x 250 bp paired-end sequencing using the Illumina MiSeq platform
  2. Data output: 12,000 contigs (> 500 bp) including reads from the pest insect and Sulcia muelleri; MIRA and Velvet assembly revealed two large scaffolds for Nasuia (~102 kb & ~12 kb)
  3. Bioinformatics: Many tools have been used, including Velvet for inital read assembly; SOAP2 to map the symbion-derived reads to the Velvet contigs and MIRA for re-assembly of isolated symbiont reads

The biggest challenge with this genome mixture was definitely the bioinformatic analysis. During several cycles of mapping and assembly the reads that belong to one organism needs to be filtered out of the remaining reads. But this labour-intensive approach revealed the smallest bacterial genome yet sequenced (112 kb).

Read the complete publication here.

Whose Genome Has Been Sequenced? – Recent posts:

News From The World Of NGS

Pacific Bioscience’s instrument PacBio RS is known as the third generation sequencing technology. And again they proof their innovative character. During the course of the last couple of months two new chemistry packages have been released (XL & P4). And during the last couple of days two even more interesting news were spread:

1. Roche and PacBio’s signed an agreement for codeveloping diagnostic products for the PacBio RS instrument (Genome Web). From my point of view this is a huge signal. Roche, as an experienced player in the NGS market with its own sequencing instruments see’s a lot of potential in the SMRT technology. So the PacBio RS system obviously got out of the teething phase and will increase its importance in the NGS business in the coming months.

2. And also New England Biolabs a big player in the area of enzyme production, proteomics and drug discovery is using the PacBio RS to study bacterial methylomes and work on new reagents for 5-mc detection. And the CSO from NEB highlights that they have choosen “the PacBio system to study bacterial methylomes because of a unique feature of SMRT sequencing that enables the detection of base modifications through the system’s kinetics”. (Genome Web)

Beside the great news for Pacific Biosciences als Life Technologies or better Thermo Fisher signed a great deal with the Chinese Dx firm iGenomics to install 32 Ion Proton sequences in 2013 (Genome Web).

And clearly all developments focus on Molecular Diagnotics and Clinical Diagnostics. And to add the missing link in this news update also Illumina recently announced that they partner with G3 to identify novel biomarkers and pathways in cardiovascular disease.

Recently Launched Tools for Genomic Sequencing

Costs for DNA sequencing decreased tremendously the last years. New technologies and better methods cause that rapid drop in prices.
On the other hand, the field of sequencing is pushed forward with

  • methods to enrich nucleic acid samples,
  • kits that simplify library preparation from a variety of samples, and
  • services to assist the researcher with all aspects of sequencing.

Read more at the Nature Product Focus; the article was published in Nature 26 September 2013

Whose genome has been sequenced? Thunnus orientalis

de-novo-sequencingTalking about sealife everyone knows how sharks or whales look like or how they behave. Sadly, I think little is known about tuna. Tuna is more or less only known as delicous meal. So it’s all the more pleasant to see that the recent de novo genome sequencing approach of Nakamuar et. al aim to learn more about the predatory behaviour of tuna and not about breeding or cultiviation (Nakamuar et. al). With this genome sequencing project of Thunnus orientalis the scientists could prove that tuna harbors some unique tactics to catch their prey.

What was sequenced?

The diploid genome of a wild-caught male Pacific bluefin tuna (T. orientalis) was sequenced.

Sequencing strategy: Whole genome sequencing

  1. Hybrid approach: Roche 454 GS FLX Titanium & Illumina GAIIx
  2. Libraries: Shotgun & paired-end libraries on Roche 454 & paired-end libraries on Illumina GAIIx
  3. Read output: 31.9 million 454 reads, including 4.9 million long paired-end reads (11.9x coverage) & 229.7 million Illumina paired-end reads (43x coverage)
  4. Data output: 192,169 contigs (> 500 bp) that could be assembled in 16,802 scaffolds (> 2 kb), totaling 740.3 Mb (= 92.5% of the estimated genome size (~ 800 Mb))
  5. Bioinformatics: Roche 454 read assembly with Newbler (Version 2.5) followed by mapping of the paired-end Illumina reads with Bowtie (Version 0.12.7).
    Note: 7,259 nucleotide mismatches & 312,851 short InDel’s could be eliminated by mapping the Illumina reads onto the scaffolds by bwa (Version 0.5.9)

Sequencing strategy: Transcriptome analysis

  1. Libraries & Sequencing: Normalized cDNA libraries have been sequenced with the Roche 454 FLX Titanium Instrument
  2. Read output: 3.8 million 454 reads
  3. Data output: 5,741 full-length cDNA sequences
  4. Bioinformatics:Assembly was performed using Newbler (Version 2.5)

From the sequencing strategy point of view this publication shows again that the hybrid approach of the Roche 454 long read technology and the Illumina short read technology is one of the most used techniques for de novo genome sequencing (Hybrid assemblies).

From a scientific point of view this publication could show that tuna hs the most RH2 paralogs among studied fishes and that three of these genes are mutated compared to the others. And according to Nakamuar et. al these changes might be responsible for the great feature of tuna to detect blue-green contrasts and therefore to be able to measure the distance to prey in the blue-pelagic ocean.

Read the complete publication here.

Whose Genome Has Been Sequenced? – Recent posts:

Running a NGS Company Is Profitable

A recent article in GEN (Genetic Engineering & Biotechnolgy News) ranked the 2012 salaries of the TOP 20 CEO’s of life sciences tools and technologies providers.

Amongst them: the CEO’s of PacificBiosciences, Illumina, Life Technologies:

Michael W. Hunkapiller, Ph.D. (PacBio) $2,177,002

Jay T. Flatley (Illumina)  $8,171,080

Gregory T. Lucier (LifeTech) $10,268,445

But although this is really impressive, it’s still not outrageous. Imagine being a 28-year old soccer player: you can earn 4times as much as G.T. Lucier … (Forbes)

The complete article can be found here.

Interview With Dr. Georg Gradl About Genome Sequencing

georg_gradl_lowBehind the scene…

You may know that Floragenex and Eurofins MWG Operon launched a new partnership to promote RAD sequencing.

As part of Floragenex’ series of interviews on genomic applications, they talked to our colleague and NGS expert Dr. Georg Gradl about his experiences with de novo genomic sequencing.

Check out the interview

GENReport: NGS Trends In Cancer Research & Clinics

GENReport_Publication growth
Enal Razvi, Ph.D. from SelectBio U.S. summarised very nicely the trends of next generation sequencing in cancer research and cancer diagnostics for Genetic Engineering & Biotechnology News.

Visit the GEN website to download the complete PDF report.

De Novo Genome Sequencing of Sunflower Via RAD-Seq

sunflowerDear NGS Expert Blog reader,

As part of our ongoing series of posts on RAD sequencing, I wanted to share some results from a recently published study describing the use of RAD-Seq for high throughput SNP development in Helianthus annuus (Sunflower).

Sunflower is one of the leading oilseed and confectionery crops in North America, with an annual crop mass of approximately 1 billion kilograms and an economic value over 720 million USD. Despite the economic importance of sunflower, relatively modest genomic resources exist for molecular genetic and marker assisted breeding applications.

To accelerate genomics resource development in sunflower, Floragenex was tasked with rapidly identifying a large set of single nucleotide polymorphism (SNP) markers in North American sunflower through the use of RAD sequencing. The end goal was to translating those markers into a downstream genotyping assay, which could be used for high-throughput applications such as linkage and association mapping.
Some highlights on this study:

  • RAD-Seq was used to rapidly construct over 15.1 Mb of de novo sunflower genomic sequence, comparable in size to a small eukaryotic transcriptome.
  • There were over 94,000 putative SNP markers identified from analysis of six sunflower lines sequenced via RAD-Seq.
  • 16,467 of these variants were incorporated into an Illumina Infinium Genotyping Array.

The above study elegantly demonstrates how RAD is an incredibly efficient marker discovery tool. From just under half a lane of Illumina data (44M 2x80bp reads), a marker resource of over 16 thousand high quality variants could be rapidly generated and deployed for breeding applications.

The full article, entitled “De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach” can be found on BMC Genomics.

As a co-author on the publication, I would be happy to answer any of your questions on this paper, so don’t hesitate to post them. For my next NGS blog entry, I’ll be showing you some interesting publication trends seen with RAD sequencing.

Rick Nipper
President, Floragenex

Whole Genome Sequencing And You

What is a genome? What are the basics of how whole genome sequencing works? What are the potential benefits and risks? Or do you just want to learn a bit more about this technology?
Ths video was developed by researchers at Mount Sinai’s Department of Genetics and Genomic Sciences and Department of Emergency Medicine with funding from the Charles Bronfman Institute for Personalized Medicine.

Just take a cool drink and relax while watching the video.