VR真人彩票

Introduction

Most of us were taught not to stick out our tongues in polite company, but this is exactly what biologist Toni Gabald贸n, PhD asked thousands of Spanish teenagers to do as part of the Saca la Lengua project.¹ Spanish for 鈥淪tick out your tongue,鈥� the goal of Saca la Lengua is to survey the oral microbiome of 15-year-olds from across Spain to learn more about the thousands of different microbes living and thriving in our mouths. Dr. Gabald贸n and his colleagues at the Centre for Genomic Regulation (CRG) in Barcelona, are using the MiSeq System to sequence more than 1600 oral samples provided by high school students throughout Spain.

鈥淪aca la Lengua was a good project to showcase the science that we do at CRG,鈥� Dr. Gabald贸n said. 鈥淢icrobiome research is an emerging field that鈥檚 expanding quickly. New discoveries are being made every day linking the microbiome and human disease, and how our diet and lifestyles might influence the microbiome. For these discoveries, you need advanced sequencing technology and bioinformatics tools to analyze the data. Thanks to technology developments such as next-generation sequencing (NGS) systems such as the MiSeq and HiSeq Systems, we can now assess the diversity of microbes that live in and on our bodies faster and less expensively. That was not possible until a few years ago.鈥�

Saca la Lengua is more than a straight microbial genetics project. It combines citizen science with community outreach to spark the next generation鈥檚 interest in science. While data analysis isn鈥檛 yet complete, Dr. Gabald贸n and his colleagues have already begun making important and surprising discoveries about the oral microbiome.

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Studying the fungal microbiome

In the past few years, microbiome research has become a hot topic. However, not all aspects of this new research area are flourishing. Research studies of the bacterial microbiome of the gut and skin have dominated the field. Fungal microbiome studies have lagged, due to the difficulty in culturing fungi and the resulting lack of robust genome databases. They also often comprise a small percentage of the overall microbiome. While their concentrations might be small, the presence of fungi in our oral microbiome can have a profound impact.

鈥淭hink of the mouth microbiome as an ecosystem just like that of the African savannah,鈥� Dr. Gabald贸n said. 鈥淭here might be very few lions compared to zebras, but if you remove the lions, you alter the entire ecosystem. Fungi play a similar role. Some fungi produce antimicrobial compounds; others produce compounds that facilitate bacterial colonization. Fungi consume oxygen, change pH, and otherwise alter the ecosystem in the mouth. When the bacteria Streptococcus mutans, one of the species that causes dental caries, grows in concert with the yeast Candida albicans, a positive feedback loop is created that causes cavities to grow larger and faster.鈥�

Fungal genomics is more difficult to study than bacterial genomics. To sequence the bacterial genome, scientists have developed universal markers that amplify given regions, with these markers working across many different bacterial species in a similar way. In contrast, it鈥檚 hard to break open the thick fungal cell walls to extract and sequence DNA. Existing primers and other tools also tend not to work as well in fungi as they do in bacteria.

As a result, there is almost no information available about the variety of fungi that live in the human mouth. 鈥淭he few studies that are available are based on small sample studies of people with certain diseases,鈥� Dr. Gabald贸n stated. 鈥淭here are no studies in young populations. Saca la Lengua project provided us with a valuable opportunity to fill this important information gap about the fungal microbiome.鈥�

鈥淭he MiSeq System provided us with a winning combination of high-throughput sequencing and long reads.鈥�

Saca la Lengua hits the road

Because so little is known about fungi in the oral microbiome, Dr听Gabald贸n suggested that CRG Director Luis Serrano, PhD, and the sequencing core at the Centre for Genomic Regulation include these organisms in the Saca la Lengua project. They readily agreed. With funding from La Caixa Bank, Dr. Gabald贸n and his colleagues put out a call to teachers in high schools

throughout Spain. 鈥淭hey focused on 15-year-olds in particular because this is the age when many Spanish teens are deciding whether to focus on science or another subject for their high school degree. Even if participating in the project didn鈥檛 encourage some of these teens to pursue science, it would improve their scientific literacy.

Focusing on such a narrow age range had an additional benefit: it made it easier for the researchers to compare differences in the oralmicrobiome. 鈥淭hese young people aren鈥檛 recruited from hospitals, but from high schools,鈥� Dr. Gabald贸n said. 鈥淭heir incidence ofdiseases such as asthma, reflect what鈥檚 found in the normal population. We want to describe the microbiome in this population and how it varies with different factors.鈥�

鈥淔rom a research perspective, the Saca la Lengua project has obtained an excellent set of normal samples,鈥� Dr. Gabald贸n added. 鈥淭hey鈥檙e all from the same regional area in an age-controlled population. If you look at similar studies, you won鈥檛 find one with as many samples.鈥�

Besides providing a sample of their mouth microbes, the more than 1600 Spanish teens also answered a series of questions about their diets, hygiene, and lifestyles. The researchers want to compare the effects of these variables on the microbiome, but also the effects of the family鈥檚 socioeconomic status, and whether the adolescents live in an urban or rural area.

MiSeq System becomes a sequencing workhorse

The CRG team relied on the MiSeq System to perform the sequencing work. They waited until they had collected all the samples, before performing DNA extractions. They then pooled barcoded samples in the same MiSeq flow cell lane to optimize the number of sequencing runs and the amount of information obtained per sample. Rather than using a whole-genome shotgun approach, the Saca la Lengua team used a targeted approach, amplifying only the region that encodes ribosomal RNA. In bacteria, they looked at 16S rRNA and in fungi, 18S rRNA. These regions provide valuable information about the identity of microbial species. 鈥淭he MiSeq System provided us with a winning combination of high-throughput sequencing and long reads,鈥� Dr.听Gabald贸n said. 鈥淲e performed previous studies with the Roche 454 and the throughput wasn鈥檛 even close to what we obtained with the MiSeq System. The reads were also long enough to overlap the DNA region between the two primers flanking the ribosomal RNA sequence, enabling amplification of the entire region of interest.鈥�

The primers used by Dr. Gabaldon鈥檚 team to target the ribosomal RNA gene also include VR真人彩票 functional adapters, providing a seamless transition between library preparation and sequencing. The completed libraries were then sent directly to the CRG core lab for sequencing on the MiSeq System. 鈥淲e鈥檙e interested in sharing what we鈥檝e learned,鈥� Dr. Gabald贸n added. 鈥淭he protocol we used is available to scientists who are interested in performing this type of study.鈥�

In addition to the MiSeq System, the team uses the HiSeq System for resequencing fungal genomes and for de novo sequencing. 鈥淔rom the genomes, we prepare paired-end and mate-pair libraries and also perform RNA-Seq,鈥� Dr. Gabald贸n said.

Where are the fungi?

Shortly after they began sequencing, Dr. Gabald贸n and his team realized that many of their samples didn鈥檛 show any signs of fungi. They cultured some of the samples and double-checked the sequencing results against samples from CRG staff who were over the age of 30. It verified that low concentrations, or the complete absence of fungi, was characteristic of the oral microbiomes of this teenage population. 鈥淲e were not expecting this result and it might have some implications,鈥� said Dr.听Gabald贸n. 鈥淚t might be that fungi colonize our oral microbiomes later in life. We鈥檙e not sure if there are correlations with dietary habits. It鈥檚 too early to say.鈥�

鈥淲ith VR真人彩票 sequencing systems, I can perform whole-genome sequencing, or by altering a few conditions, perform RNA-Seq and obtain a large amount of data to answer a genomics question.鈥�

Aside from the surprising absence of fungi, sequencing of the samples has been easy and straightforward. The data analysis portion of the project, has been more complicated. The Saca la Lengua team obtained millions of reads per sample, representing the rRNA gene region in different bacterial species. That amount of data presents a challenge in determining which bacterial or fungal species or genera are present in each sample. Dr. Gabald贸n used an analysis pipeline that mapped the MiSeq reads to a database and used a scoring system that measured the amount of alignment and the similarity of sequences. With these scores, the researchers could say whether they had Streptococcus mutans or another Streptococcus species.

A better understanding of the oral microbiome

鈥淲e are very happy with the quality of the MiSeq data,鈥� Dr. Gabald贸n said. 鈥淎ny problems that we鈥檝e had have been generally due to the quality of the DNA.鈥� The MiSeq System has also opened a door to understanding the oral microbiome. 鈥淲ith VR真人彩票 sequencing systems, I can perform whole-genome sequencing, or by altering a few conditions, perform RNA-Seq and obtain a large amount of data to answer a genomics question,鈥� Dr. Gabald贸n added. 鈥淎 few years ago, that was impossible. I had to rely on publicly available sequencing data from large consortia for certain genomes. With the VR真人彩票 systems, I can be proactive and if I need a certain genome, I can sequence it myself.鈥�

Data analysis has just begun and Dr. Gabald贸n doesn鈥檛 yet have information on the bacterial and fungal species they found and what amounts, or how the oral microbiome might be related to lifestyle and health variables. That hasn鈥檛 stopped other researchers from taking an interest in the project. 鈥淚鈥檝e received interest from researchers to expand the project and initiate studies in their countries,鈥� Dr. Gabald贸n said.

Connecting with the citizenry

An unexpected benefit of the Saca la Lengua project has been the ability to connect with teachers and students. 鈥淭his is the first time I have been involved in a project that has brought me so close to the citizens of Spain,鈥� Dr. Gabald贸n said. 鈥淚鈥檝e met with teachers and students participating in the project, discussing it with them and answering their questions. Giving a talk in front of an audience of 15-year-old students is harder than talking about your research with a group of scientists at a meeting. You need to make a significant effort to connect with them.鈥�

Moving forward

Dr. Gabald贸n hopes that the sequencing data and metagenomics studies of these specimens will enable him and other researchers to learn more about the normal, healthy oral microbiome, and how assorted variables can alter the make-up of mouth microbes. These insights, in turn, might provide more information about various chronic diseases that affect teens and adults. 鈥淎s we learn more, we are realizing the microbiome is connected with diseases such as Crohn鈥檚 disease, and some diseases that you might not even think of, such as arteriosclerosis,鈥� Dr. Gabald贸n said. 鈥淚n principle, it seems odd that there might be a connection. Yet, every time you brush your teeth there are some cells that enter the blood stream. Some of them are able to trigger a significant immune reaction that might create granules in the blood that could cause veins to collapse, for example. It all depends on which bacterial species you have in the mouth. We are learning that the microbiome is a factor that influences our health status and might be a factor in some important diseases.鈥�

He also points out that the oral microbiome has advantages with sample collection, because a saliva sample is far easier to obtain than a biopsy. 鈥淭he more scientists understand the oral microbiome, the more research opportunities open up to improve human health,鈥滵r. Gabald贸n added.

Learn more about the VR真人彩票 system mentioned in this article:

MiSeq System, www.illumina.com/systems/sequencing-platforms/miseq.html

References

1. Saca la Lengua. . Accessed March 31, 2016.