Tag Archives: metagenome

Don’t forget the controls!

Almost every day new data about the composition of microbiomes are published. Many of these studies analyse the human microbiome, but also environmental samples.

Today we have the ability to sequence microbiomes in much more depth than a couple of years ago. Looking deeper sheds light on an important point: Contamination! In the very interesting publication of Salter et al. they could show that contaminating DNA is present in DNA extraction kits and other lab reagents.

The researchers sent dilutions of pure cultures of Salmonella bongori to three different institutes for DNA extraction and PCR, followed by sequencing on Illumina MiSeq. While S. bongori was the only organism identified in the undiluted samples, contaminating bacteria increased in relative abundance with higher degrees of dilution, and finally became dominant after the fifth dilution.

They did a similar analysis performing shotgun metagenomics of a pure S. bongori culture. This time, they used four different DNA extraction kits. Again, they saw that contamination increased with the degree of dilution, with contamination being the predominant feature after the fourth dilution. Also, they could show that each kit gave a different bacterial profile.

They also report on a study on the nasopharyngeal microbiota of children, analyzed over 2 years. They could show that using 4 different DNA extraction kits over time led to the false conclusion that differences in the microbial spectrum were associated with age. When DNA extraction was repeated on original samples using a different kit lot, the OTUs previously identified as contaminants were no longer detected.

In conclusion, contamination affected both 16S and metagenomic shotgun sequencing projects and was especially critical for samples with low biomass. Salter et al. present a list of potential contaminating organisms, as well as recommendations on how to cope with this problem. One recommendation is very obvious, and very effective: use negative controls!

Altogether, we should be very careful in planning our experiments in order to deliver results instead of artefacts. Especially, we need to be very careful when interpreting the data!

Think Big: American Gut Project Based On NGS

Scientists estimate that the cells of our bodies are outnumbered 10 to 1 by bacterial cells which live in or on our body.  A previous blog has already pointed out the impact of this fact on sequencing the corresponding host genomes. On the other hand, microbiomes have the potential to play an important role as diagnostic markers, or opening up new ways of treating diseases, such as personalized medicine.

However, we are just beginning to understand the complex relationships of this “social network”, as the Scientific American has called it. The most complex bacterial community within the human body resides inside the gut. In order to obtain a deeper understanding of the bacterial communities of the human gut, there have been several attempts of sequencing the gut microbiomes of larger groups of individuals, such as projects by Arumugam et al., Yatsunenko et al or Schloissnig et al. However, so far, the number of individuals which were analyzed was relatively small (up to several hundreds).

A group of US scientists have now started the “American Gut Project“.  As reported by Genome Web News, this project is planned as a crowd-sourcing study of 10.000 or more individuals in the US. Since this study is part of the “American Food Project”, it will mainly focus on gut microbiome patterns in relation to diet, age and lifestyle. People who would like to participate in this study need to sign up via a website and donate $99. This money will be used to cover a significant part of the cost of the study. In return, participants will receive a taxonomic profile of their gut microbiome.

The analysis itself will be based on 16S sequencing. For part of the samples, additional analyses such as sequencing the complete metagenomes and long term surveys are planned. No doubt, this study will clearly provide us with a huge data set. However, this data set will be highly complex. Also, it still needs to be brought in context with data from other projects.  To my opinion, interpretation of the data still remains the hardest part. Or, as project organizer Jeff Leach has put it in an interview with Genome Web Daily News: “We don’t expect to be able to address some questions, but because of the size of the sample and because of the broad patterns we expect to see in diet and lifestyle, we think some stuff will fall out.”

Sequencing than soaking in Hot Spring

There are many volcanoes and earthquakes in Japan, but it is not always a bad thing, they are also responsible for the many hot springs. Most Japanese people love soaking in a hot spring and they believe that this eliminates fatigue and improves health. Hot springs also had a great contribution to biotechnology via the heat resistant DNA polymerase from Thermus aquaticus (Taq) and its derivatives. Not only PCR, but also Sanger sequencing was accelerated by these heat resistant enzymes as we all know well.

Scientists have started to study the genome/transcriptome world in hot springs with NGS technologies. Murakami et al., peformed 16S-rRNA (Sanger sequencing) and meta-transcriptome analysis from small RNA (GS FLX sequencing) of groundwater (up to 1,000 m depth) from Yunohara hot spring, Japan. Their phylogenetic analysis using 16S rRNA showed the classification of 17 species including archaea and eubacteria.  There are only 2 or 3 dominant species in typical cases of other hot springs, but this one is rich in diversity. Furthermore, they found the very unique group “Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN)” which is a small organism/cell with only 200 nm size! Their small RNA analysis identified 64,194 (20,057 nonredundant) cDNA sequences, and they found several novel non coding RNAs which have a very stable secondary structure.

Therefore, hot springs may still be gold mines for useful genes and important biological knowledge of unknown underground ecosystems.



Metagenomic Assembly: The Big Challenge

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.

What Is Your Gut Microbiome Type?

A social network wants to sequence your gut microbiome. The non-profit programme MyMicrobes, is inviting people to have their gut bacteria sequenced for about €1,500. Acting as both, social network and DNA database, the website offers a place for people to share diet tips, stories and gastrointestinal woes with one another. In exchange, researchers hope to gather a wealth of data about the bacteria living in peoples guts.

The same team of researchers showed earlier this year that people fall into one of three groups, or enterotypes, when it comes to the genetics of their gut bacteria (Gut study divides people into three types).

So far, the team has found links between certain gut-specific genetic markers and obesity and other diseases. And they suspect that gut enterotypes might affect how people react to different drugs and diets.

The group has been careful not to make promises to project participants. The research is still in its infancy, and there is no proven link between enterotypes and disease remedies. But the researchers hope the project will provide a bounty of data while helping volunteers connect with one another. Participants will have access to their own data, but all public results will be anonymous.

What is your opinion? Are you ready to sequence your gut microbiome?

Is 16S rRNA Sequencing Ready for Illumina Sequencer?

Claesson et al (2010) compared for the first time 16S rRNA metagenome sequencing on Roche GS FLX and Illumina GAIIx. Several tandem combinations of variable regions were targeted in microbial DNA extracted from fecal sample material and sequencing of the PCR amplicons was performed in a quarter segment of a full GS FLX run and a full lane of GAIIx with 2x 100 bp module.

Comparison revealed that taxonomic classification down to genus level is a lot worse for Illumina reads because of their shorter read length and higher error rate. Also the insight into rarely occurring species is far from increasing considerably in comparison to GS FLX sequencing, as it might have been expected because of the significantly higher coverage.

As the authors suggest a big step forward to increase classification efficiencies with Illumina technology is sequencing of fragments with insert sizes smaller than 200 bp. Following this strategy forward and reverse read which display poor quality at the read ends are overlapping and thus generate a consensus sequence with improved quality.

For sure respective studies are underway and we will continue to monitor the latest developments concerning this topic.