In the NGS (Next Generation Sequencing) group of the networking platform LinkedIn I came across a lively discussion about the best strategy for de novo sequencing of a bacterial genome. The discussion was about technology (Roche versus Illumina), coverage (from 10-fold on Roche GS FLX to 500-fold on Illumina HiSeq 2000) and library types. The comments and advices given coincide with our experience that this question can not be answered without any further information on the genome to be sequenced. GC content, amount and size of repeat structures as well as the genome size of the bacterium have to be considered. We have meanwhile de novo sequenced more than 100 microbial genomes and according to our experience GS FLX technology with a combination of shotgun and long paired end libraries will deliver a high quality genome sequence that is suitable for gap closing projects.
Are you interested in refreshing your knowledge on the Illumina sequencing technology?
Enjoy the animation that very well illustrates the sequencing of a genomic DNA sample on Illumina platforms. After fragmentation and ligation of adaptors fragments are amplified on a solid surface via bridge PCR. For sequencing reversible terminators are applied in a cyclic way comprising nucleotide incorporation, imaging and cleavage of the blocking group (cyclic reversible termination). Contrary to the way it is presented, the sequencing primer is binding to the upper part of the template and strand elongation is done downwards.
In 2008 two research groups have independently shown in proof of concept publications that trisomy 21 can be detected by high-throughput sequencing of cell free fetal DNA from the mother’s blood plasma with high specificity and high level of sensitivity (Fan et al., 2008; Chiu et al., 2008).
In short, Illumina reads are mapped to the reference human genome and by counting the reads that map to each chromosome the relative dosage of each chromosome is measured. In case of trisomy 21 or other aneuploidies the relative dosage of the affected chromosome is statistically overrepresented in the data set.
Within the last two months the companies Sequenom (Ehrich et al., 2011) and Verinata Health (Sehnert et al., 2011) both have published studies examining large test sets of patients. Both companies apply sequencing on GAIIx. The approaches mostly differ in normalization of sequencing data and bioinformatics analysis. Using a smart algorithm for normalizing the chromosome-counting data Verinata Health was able to classify 100 % correct all trisomy 21 and trisomy 18 samples in the test set (Sehnert et al., 2011).
While Sequenom recently finished the Clinical Validation Studies (GenomeWeb and Sequenom’s Web Page), Verinata Health is currently conducting a blinded clinical study to further demonstrate the diagnostic accuracy of their method (Sehnert et al., 2011)
Current screening methods are usually showing high false-positive results. Only invasive methods with high risk for mother and fetus provide definite genetic information about the fetus. The NGS based detection of fetal aneuploidy in high-risk-pregnancies is very promising from my point of view, as the method is non-invasive and is so far displaying high sensitivity and specificity.
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Eurofins MWG Operon is organizing NGS roadshows throughout Germany. After our visits to the Bavarian cities Würzburg and Erlangen the next stops on the tour will be Hamburg (May 24th), Kiel (May 25th) and Berlin (May 26th). On June 20th, we are inviting to our roadshow in Freising (near Munich). In Baden-Württemberg we are looking forward to welcoming you on June 7th to Tübingen, on July 11th to Heidelberg and on July 12th to Freiburg.
Core of the roadshows are presentations on NGS technologies and examples with a focus on latest developments. Subsequently interested parties have the opportunity to discuss projects in face-to-face conversations with our NGS experts.
For more information please see the official invitations.
Next generation sequencing allows researchers to generate high resolution maps of epigenetic modifications for whole genomes (epigenome). Whole genome methylation pattern has been determined e.g. for A. thaliana by sequencing bisulfite treated DNA with Illumina technology (Lister et al., 2008 and Cokus et al., 2008).
In the near future epigenetic studies may be possible without bisulfite treatment by single-molecule real-time sequencing (SMRT). The first proof of concept study promises direct detection of modified nucleotides as well as discrimination between different types of modifications (Flusberg et al., 2010).
Six and five years after the commercial launch of their first next generation sequencers, the companies Roche and Illumina are still at the forefront of the next generation sequencing market. By today, Roche technology has enabled 1209 peer-reviewed publications while Illumina technology has even enabled 1631 publications (from both companies’ websites).
The current sequencer of both companies are the Roche GS FLX with an average read length of 350-450 bp and 400 Mbp data output per run and the Illumina HiSeq 2000 with read length of up to 100 bp and up to 320 Gbp data output per run.
Both companies’ focus on technological improvements is mainly on enhancing read number and read length. Illumina has recently launched the 150 bp read length (for GAIIx only) and has further announced doubling of yield for Q2 in 2011. Roche is planning to launch the extended average read length of 750 bp sometimes before the end of June according to GenomeWeb News on April 19th, 2011.
In my point of view third generation sequencing will not replace these technologies but enable additional applications as well as combining third generation and second generation technologies for specific project layouts.
Great retro webcast: Nobel laureate James Watson and former NHGRI Director Francis Collins discuss the history and future of human genetics. It was created in 2003 when the human genome was sequenced the first time. In 2003 the 50th anniversary of James Watson and Francis Crick’s Nobel Prize winning description of the DNA double helix was also celebrated.