NGS Expert Blog

Photographer: Florian Gerlach from Nawaro

Does read length really matter when doing a de novo assembly of BACs from highly repetitive plant genomes, like that of barley? Will paired end sequencing improve the assembly considerably? Is it essential to barcode each BAC clone before sequencing? 

These are the main questions of a study from Taudien et al, published in September 2011, answered by comparing assembly data derived from different sequencing data sets.

To investigate the effect of the read length the authors compared the assembly quality from 4 BAC clones with either FLX or with Titanium chemistry reads at equal sequencing depth. Even though the average read length differed by only 40 bp (223 vs 263 bp) the assemblies with the longer reads were considerably better. They contained fewer missassemblies and reduced number of gaps. According to the authors this is mainly due to the broader read distribution of the Titanium chemistry, where a fraction of reads displayed a read length of >600 bp.

These findings are in accordance with our experience from BAC assemblies. Having performed several BAC sequencing projects with GS FLX+ yet (average read length of 550-650 bp and some reads up to 1000 bp), we can report here that FLX+ read length has the potential to even more improve the BAC assembly quality. 

Please have a look at the research article for further information.

With today’s post we would like to present to you Mr. Jay T. Flatley, CEO of Illumina since 1999. His view of costs and markets in the area of Next Generation  Sequencing is giving a real good insight into the market development.

>> Watch the video

Very recently researchers from Stanford University systematically investigated performance of the most widely used exome enrichment platforms:

1. Roche/NimbleGen’s Seq Cap EZ Exome Library v2.0 (44 Mbp)
2. Agilent’s Sure Select Human All Exon (50 Mbp)
3. Illumina’s Tru Seq Exome (61 Mbp)

One of the findings of the study is: When comparing coverage efficiency at constant read depth (80 million reads each) NimbleGen Sequence capture is by far better than the other two platforms. With NimbleGen sequence capture 98.6 % of all targeted bases were covered at least 10x, while Agilent’s Sure Select and Illumina’s Tru Seq covered only 89.6 % and 90.0 % of all bases at least 10x. In my opinion, the different target sizes of the exomes should have been taken into account. In this case the read depth should have been normalized according to the exome sizes. Independent of the missing normalisation it is however clearly shown in the paper that the NimbleGen technology enriched a much higher percentage of the targeted bases than the other two products..

Other criteria that were compared are the off-target enrichment rate (NimbleGen performed best) as well as the enrichment bias owing to GC content (Agilent performed best).

The decision, which platform is best for a specific scientific question should also be influenced by the individual target regions covered by different Exome kits. Agilent’s and NimbleGen’s exomes share 38 Mbp of their target regions. Apart from that Agilent’s Exome covers better Ensembl genes, while NimbleGen’s Exome covers a greater portion of miRNAs. Illumina’s exome, although displaying low coverage efficiency, is designed to capture UTRs in addition, which by now are almost not covered by the other designs and is therefore the choice, if those regions are of interest.

Differences in the performance come from the different oligonucleotide designs. I therefore postulate similar key parameters when using the customised versions of the capture technologies.

Recently Illumina reported a drop in preliminary Q3 revenues. They expect 1% less revenue compared to previous Q3, while Wall Street expected 17% growth (genomeweb). Illumina explains this with uncertainty in funding and higher throughput of the V3 sequencing Kit. They also report that the upgrade rate from Genome Analyzer to HiSeq 2000 was lower as expected and that the use of Genome Analyzer reagents dropped significantly.
In an earlier post I have already speculated that many sequencer are only used at a low percentage of their capacity. This might explain the low upgrade rate of older sequencer and the drop in use of reagents. They are simply not in use.
I have currently no information about the upgrade rate of Roche GS FLX/454 sequencer for use of the new FLX+ chemistry.
Did you upgrade your sequencer already?

Eurofins MWG Operon and Integrated Genomics have announced a cooperation agreement to combine their expertise in sequencing and analysis services for microbial, fungal and algal organisms. The goal of the cooperation is to provide customers “one-stop-shopping” for complete genome projects that delivers analysis results from the raw extracted DNA. Read the press release.

Chinese hamster ovary (CHO) derived cell lines are currently the most popular expression systems for biopharmaceutical protein production, including monoclonal antibodies, growth factors, hormones and enzymes. CHO cell lines have gained this dominant role, because their enzyme sets perform human-compatible post translational modifications and because protocols for transfection and clone selection are well established.

Recently, the draft genome sequence of the CHO-K1 cell line was published in Nature Biotechnology. The Illumina sequencing data have been assembled to a 2.45 Gbp draft genome. The sequence will probably facilitate CHO-cell line engineering to improve yield and quality of protein production.

However, having performed non-targeted insertion of constructs in eukaryotic cell lines (like CHO-cells) or organisms, or studying virus integration in eukaryotes requires a method for analysis of the integration sites and quality and copy number of the insert. Primer walking on genomic DNA is very problematic and therefore does not provide the answer.

Therefore we currently develop an assay for easy and straight forward analysis of insertions sites and the inserted sequences. First test projects are running….

We are optimistic that we can inform you soon about the outcome.