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)

With this new bi-weekly series we would like to highlight some if not all genomes that have been sequenced in the last 6 to 12 months. And at this point of time I am still uncertain if the diversity of organisms and species will be the “eye-opener” or the different technologies and strategies that have been used…

We started this series off in January where we reported about the de novo sequencing of the domestic goat Capra hircus.

Today I would like to report about a plant genome, the Cicer arietinum:

According to the GenomeWeb article this de novo genome sequencing approach is only the 3rd one for crop legume plants. For me that is kind of astonishing since breeding and optimisation of crop is already done since years. Maybe this is due to the huge genomes of plants that outperform animal genomes by far. For our chickpea plant with 740 million base pairs we talk about a medium size plant genome. But let’s focus on the sequencing approach for now (Varshney et. al):

What was sequenced?

De novo sequencing of one reference chickpea plant and re-sequencing of 90 cultivated & wild chickpea lines from 10 different countries

Sequencing strategy:

1. De novo genome sequencing on HiSeq 2000 (paired-end module) of 1 genome with 11 shotgun and mate-pair libraries (insert sizes: ~ 170; 500; 800; 2,000; 5,000; 10,000; 20,000 bp) and BAC end sequencing
   Data output: 153.01 Gb; after filtering & correction steps only 87.65 Gb data were used for de novo assembly
2. Re-sequencing of genomes
   • Whole genome re-sequencing on 29 varieties using Illumina 100 bp paired-end sequencing on HiSeq 2000
   • RAD-sequencing of 61 genotypes on HiSeq 2000 (48x ApeKI; 24x HindIII)

According to D. Cook “the sequencing of the chickpea provides genetic information that will help plant breeders develop highly productive chickpea varieties that can better tolerate drought and resist disease — traits that are particularly important in light of the threat of global climate change”. (Davis Enterprise).

Read the complete publication here.

The High-Throughput Sequencing map by James Hadfield (Cancer Research UK, Cambridge) gives us a very interesting overview about sequencing activities around the world. We ran a survey to find out if your favourite machines correspond with the platforms listed by James in his overview.

Here are the results: Your personal favourites are nearly a perfect match with platforms in the genome centers worldwide. Great match!

30 years of PCR in various applications has revolutionised molecular biology. But PCR also has its drawbacks. One of them is the amplification of AT- or GC-rich DNA fragments. Naturally, researchers are often interested in sequencing and studying genomes with high GC or high AT content, like S. aureus with a AT content of 67% or Streptomyces coelicolor with a GC content of 72%.
But more and more NGS kit providers try to circumvent PCR in the library prep. Ashley Yeager has summarised the current status of PCR-free library preps including a comprehensive overview of the pro’s and con’s of both methods (BioTechniques).

Summarising the findings from Mrs. Yeager there is no clear champion in sight:

Library prep by using PCR methods
+ well-known lab procedure & good sequencing efficiency
- difficulties in amplifying GC- / AT-rich regions -> sequencing is biased

PCR-free library prep
+ good sequence read distribution & a more even genome coverage
- huge amounts of starting material needed & sequencing reaction is less efficient

Read the complete article under BioTechniques.

Newts have an extraordinary ability to regenerate tissues. For example, they can re-grow fully functional limbs after amputation. In addition, regeneration of parts of the central nervous system, the heart, and the lens has been characterized, making them an excellent model organism for studying regenerative processes. However, because of their enormous genome size (10 times that of human), the molecular mechanisms behind this amazing regenerative process are largely unknown.

A research group at the Max Plank Institute recently published a de novo assembly of the transcriptome of the urodelian amphibian Notophthalmus viridescens (Looso M. et al. ). The researchers combined 454, Illumina, and Sanger sequencing data from both normalized and non-normalized cDNA libraries. The resulted transcriptome comprises over 120,000 non-redundant transcripts. Homology search using BLAST led to annotation of 38,000 transcripts. Importantly, they found 800 transcripts, whose protein-coding potential was validated by mass spectrometry, that show no similarity to any know transcripts or show similarity to urodele-specific EST sequences. Some of these transcripts belong to novel protein families.

It is an interesting hypothesis that some of those newt-specific proteins may provide mechanistic insights into regeneration processes unique to these animals. Their work will definitely be an important resource for subsequent studies in tissue regeneration and may benefit future research in regenerative medicine.

With today’s press release I am happy to announce that AROS Applied Biotechnolgy A/S  is now a member of the Eurofins group.

Here is a short introduction of our new colleagues from AROS:

• AROS was founded in the year 2000
• AROS started as a spin off of from the Aarhus University Hospital and was the first service provider for Affymetrix in Europe
• AROS is based in Denmark and provides a long term experience in sample preparation, microarray analysis and next generation sequencing (NGS)
• Nowadays AROS has a leading position in NGS service for pharmaceutical research
• AROS is an Illumina reference lab for next generation sequencing
• The main focus in NGS is RNA-Seq and exome sequencing that is accomplished with the exome designs of the leading provider in this area (Illumina TruSeq Exome Enrichment, NimbleGen EZ Capture & Agilent SureSelect)

“AROS is an excellent fit […] with our focus on high-quality next-generation sequencing […]” (Dr. Gilles Martin) and therefore I am confident that this new alliance will help us both in further expanding our experience level in NGS and to benefit from our complementary strength.

I am sure you will hear more about the activities from AROS on our blog and hope you join me in welcoming AROS as a member of Eurofins.

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.

The analyst and sequencing community is currently divided on whether to believe the rumors of a new bid from Roche to buy Illumina. The source of the controversial discussions is an article from the Swiss Newspaper L’Agefi that reported end of December that Roche and Illumina might have agreed to a deal for Roche to acquire Illumina. Since Illumina turned down Roche’s original bid in January, continuous interest from Roche has been reported several times, but the report from L’Agefi is also mentioning concrete amounts of the bid. According to them, the acquisition might take place for $66 per share, valuing the deal at about $8.14 billion in total.

The offer is $15 per share higher than the previous offer of $51 in April last year. According to the analyst Devia Ferreiro of Oppenheimer the new bid is definitely at a level that might lead to a final deal.

With Roche having only about 9% of the NGS market and next generation sequencing becoming most likely an important clinical diagnostic tool in the next years, the strategy focus of Roche must be to get better access to the NGS market and to take NGS to clinical practice. The acquisition of the NGS market leader Illumina represents an optimal starting point.

We’ll see if the rumors are built on a solid foundation within the next two weeks: The Swiss newspaper L’Agefi reported that the announcement might come during the first half of January.

With the update of our MiSeq system to 250 bp reads genome sequencing on this system gets even more important. But long reads and huge data output are not the only prerequisite for a great de novo assembly result.

What is missing?

Paired-end libraries that span gaps and repetitive structures can improve de novo genome assemblies tremendously. Our proprietary long jumping distance libraries (LJDs) are perfectly suited for scaffolding on Illumina sequencing devices. In contrast to other paired-end libraries (like Illumina mate pair library), our LJD library preparation involves an adaptor-guided ligation of the genomic fragments. The different preparation protocol offers the following advantages:

• No hybrid reads – a unique sequence identifies the crossover points
• No shotgun pairs – less than 1% of all LJD reads are shotgun paired-end reads
• Distinct insert sizes – we prepare LJDs with 3, 8, 20 or even 40 kbp insert size
• Span large repeats – large and complex repeats up to 40 kbp can be resolved

Mapped reads: All reads from a 3 kbp LJD library (grey) are aligned to a reference sequence. Two LJD read pairs are highlighted (blue + black) and their measured insert size is 3107 bp and 3002 bp respectively.

Why should I combine MiSeq long reads and LJDs?

The new features of the MiSeq (250 bp reads; data output up to 8 Gbp) enable the combined and cost-efficient approach of shotgun and LJD libraries in one run. The MiSeq output is sufficient to sequence several bacterial genomes or single fungal genomes (up to 60 Mbp) with appropriate coverage.

• Longer reads – more sequence information to correctly map the reads onto your contigs
• Short delivery time – due to the shorter run time compared to the HiSeq 2000

Read more about our long jumping distance libraries on our website

We are happy to extend our next generation sequencing capabilities with the new Illumina HiSeq 2500 and Illumina MiSeq. Thanks to these new machines, we can provide customers with unsurpassable NGS services at high accuracy rates and shorter turnaround time (TAT). Read more at http://bit.ly/OTv9AP