NGS Expert Blog

Currently, Roche 454 has a unique selling proposition in providing the only sequencing technology on the market delivering long reads with high accuracy at the same time. Just last year Roche 454 launched the new GS FLX+ chemistry delivering reads with a modal read length of up to 700 bp. Long reads are crucial for de novo sequencing of genomes and transcriptomes and for sequencing of amplicons.

The current version of the Illumina MiSeq enables 2x 150 bp paired-end reads and 1.5 – 2 Gbp per run, which is slightly over the output of a typical GS FLX+ run. When working with short insert libraries of 200-250 bp, both paired-end reads will overlap and finally generate one longer read of up to 250 bp.

This read length is still not in a competitive range for Roche 454, but recently Illumina announced the launch of a MiSeq instrument upgrade by the middle of the year. According to Illumina ‘s vice president of marketing, the upgraded instrument will generate 2x 250 bp paired-end reads and up to 7 Gbp of data output. When sequencing short insert libraries with the 2x 250 bp paired-end module, reads with up to 450 bp can be generated. Thus, read length comes again closer to the read length of the Roche FLX+ technology (not to mention the 10x higher data output).

We have to wait and see whether the MiSeq upgrade keeps what Illumina promises, but for me personally it is quite clear, that with the announced specifications, the MiSeq will sooner or later replace Roche 454 sequencing for certain applications. In this light it is very interesting that Roche offered a friendly take over of Illumina this week.

Recently, I just discovered a study from Harvard Researchers that uses genome sequencing to investigate the genetic basis for antibiotic resistance in E. coli.

The authors used continuous culture systems that are slowly increasing the concentrations of three different antibiotics, separately. They observed that the resistance increased significantly over a period of approx. 20 days. And by sequencing the genomes of the resistant populations on GAIIx, they found in total 47 SNPs contributing to the resistances.

In trimethoprim-resistant bacteria most of the mutations occurred in the E. coli gene encoding the target enzyme that is normally inhibited by the antibiotic compound. In bacterial populations resistant to the antibiotics chloramphenicol and doxycycline, in contrast, the mutations accumulate in a broad spectrum of genes involved in translation, transcription and protein transport. This is consistent with chloramphenicol and doxycycline being inhibitors of protein synthesis.

This is of high importance since the prevalence of resistant bacteria is continuously increasing. As a result, in more and more cases, the standard antibiotics are no longer effective to treat seriously ill patients. New findings in antibacterial research are crucial to stop this progression as soon as possible.

In our latest poll that started mid of November 2011 we raised the question about your sequencing strategies for scaffolding projects. 29 ngs-expert.com readers did submit their votes.

39% of all votes agree my own opinion that LPE and LJD libraries are the preferred method for scaffolding of contigs. Long reads of up to 40 kbp can be easily and efficiently bridged.

But despite that, it is also obvious that all other techniques are still used for scaffolding projects. And I am still interested to see whether this might change with the new C2 chemistry for PacBio RS that is announced for Q1.

Microbial communities are more and more analyzed by direct sequencing of DNA from environmental samples. The aim is to study the microbial composition at different conditions and the identification of novel organisms. The bottleneck is the assembly of the metagenome reads.

Why are these assemblies so challenging? One important reason is the highly heterogeneous character of the microbial environmental sample. Furthermore the abundance of the member species differs remarkably. While some species are highly abundant, others – often the unknown and therefore very interesting ones – are present at a very low level. In order to receive contigs from the low abundant species very deep sequencing is needed and these species often can only be assembled in a highly fragmented manner. Another challenge for metagenomic assemblies are populations of closely related species. As their genomes are highly similar the assembly software generates hybrid contigs from those closely related species.

Despite or probably due to these challenges I see a lot of efforts in the field in improving the underlying assembly tools. Currently, procedures like clustering large contigs based on tetranucleotide frequency and coverage are applied. Clustered contigs are afterwards ordered by mapping to related genome. With this approach first bacterial draft genomes from e.g. cow rumen  and soil metagenomes have been published in Science and Nature.

It is well known that some people are more attractive to mosquitoes than others. In an article of the sueddeutsche zeitung I learned that this has however nothing to do with the taste of the blood, it is the composition of the skin microbiome.

Researchers around the group of the Dutch insect expert Niels Verhulst published that Anopheles gambiae, the main carrier of malaria in Africa is attracted by odours, produced by certain bacteria of the skin microbiome.

Blends produced by some bacterial species were more attractive than those of others. Test persons that had a high amount of Staphylococcus on their skin were attractant, while test persons carrying Pseudomonas aeruginosa produced odours that were repellent for the insects.

As a consequence these volatiles could be synthesized and used as mosquito attractants or repellents.

Recently we discussed already the importance of the gut microbiome. It becomes more and more obvious that we cannot regard our body as a single human being, we are part of a large community living in and on us, with all the consequences it may have.

What will come next?