quinta-feira, 29 de janeiro de 2015

Next Generation Sequencing vs Sanger Sequencing

Hi all,

Long time, no see.

We are very sorry for the long time to update the blog. We have been working like crazy since we had a big methodological change on our aproch to reach our final goal. 

Recapitulating, our main aim was to sequence the CYP1 and AHR genes of 100 Siluriformes species and this would be done by the Sanger method DNA sequencing. However we had some issues, with the primer, for instance. Basically, we were able to amplify cyp1a in just six of the 30 species we had sampled. For practical purposes, this was not good. However, it suggested that the molecular diversity of cyp1a in loricariidae fish is much greater than what we expected, which in turn is an excellent news. Our sequencing method was modified from the traditional Sanger method to one of the Next Generation Sequencing (NGS) methods.

Now we are sequencing the liver transcriptome of 40 individuals from 37 different species using the Illumina Technology Hiseq2500 (Next Generation Sequencing) at the Brazilian National Cancer Institute (INCA).

A major advantage of this new approach is that it is not based on specific primers. Now we will obtain the molecular data without the bias caused by primers. Besides, it will generate much more raw data to analyse than our previous method. Consequently, we will get the sequence of not only the two desired genes, CYP1A and AHR, but from all genes that were expressed at the time of the sampled fish liver. In fact, this method is generating more raw data than we will be able to analyse during this peer grant. During this grant period, we are focusing our attention in a particular set of genes involved in the responses of the organisms to chemical compounds. Other genes will be analysed later. Another advantage is the price per base pair which is cheaper than Sanger, as we can see in the table below.

Read Length
Up to 1.100 bases
2x 100pb
Read \Run
2 billions
1 hour

So, I will talk about the processes before and during the preparation of these libraries. First of all, we had to talk with the responsible for the Illumina Hiseq 2500 in INCA to know if it would be possible to use this sequencer, when it would happen and if the technologist, Carolina Furtado, could teach us to prepare our samples. After it was solved, we could start to work hard.

Well, this is a summary of what has being going on!

We wish you enjoy it and hope to write more often.

See you soon!

Preparing the libraries for the Next Generation Sequencing

Today let´s talk a little about Next Generation Sequencing.

After we decided for this sequencing method, we had to prepare all samples to be sequenced; which includes RNA extraction, verification of RNA quality using a method more reliable than the one we used before, construct the cDNA libraries and evaluate its quality and quantity.

Based on the nanodrop quantification, we performed another kind of analysis using the Bioanalyzer, which is a more precise equipment to assess the quality of the material that has been extracted. Using this method, a RNA Integrity Number (RIN) is generated, this is a number assigned by the software that considers also the presence of degradation products. Although Illumina recommend a RIN higher than eight for transcriptome sequencing, we sete our threshold in six due to sample particularities. By doing this, we assume the risk of getting transcriptomes biased for the 3' end of the transcripts. However, most of our samples had RIN between 7.5 - 8.0  and our first results indicates high coverture of 5' end.

Initially, we select the mRNA with special beads that contains oligo dT, this way the material will be purified and only the mRNA will stay in the well plate. After this, we fragment the RNA in a delicate step of 3 minutes at 94 ° C in the thermocycler.The longer this step takes, the more fragmented the RNA gets. This time give us fragments of about 300pb.

The next step is to synthesise the cDNA in two different reactions. First strand first and then, in another reaction, the second strand. This way we end up with double strand cDNA, and not with the hybrid cDNA used for regular molecular biology applications. When the cDNA is ready, we have to  repair their Ends to ensure that each cDNA molecule has a blunt end and contains a 5 'phosphate and an end 3'OH free.

The adapters binding step is crucial to sequencing as it is when we give an identity for each sample.  By doing this, we can sequence several different samples in the same lane. However, it is vital to take note about which adapters were used in each samples, so at the time of sequencing, we won't put samples with the same adapter in the same lane. Then, it is made a short (15 cycles) PCR enrichment of DNA fragments, where the molecules with adapters at both ends are selected and the DNA sample is amplified. In this PCR primers binds the adapters.

Finally, we need to run the libraries in bioanalyzer to check their sizes and perform qPCR for precise quantification. The size and quantity informations are important for the normalisation, when all samples are prepared to have the same number of molecules, so all the samples at the same lane will have equal chances to be sequenced. Then the samples are finally ready to be sequenced with illumina Hiseq 2500.

segunda-feira, 31 de março de 2014

Working hard on cDNA synthesis

Today, we, Maithê and Paula, will tell you a little about what we do with the collected samples of Loricariidae when they arrive in the lab. As previously written, after fishes has been collected and livers extracted, it's necessary to extract RNA and DNA. What do we do next with this genetic material?

Well, while the DNA is sent to the Paulo Buckup's team, our collaborators at the National Museum of Natural History, the RNA is used for synthesis of complementary DNA (cDNA). But what is cDNA, how is it done and serve for??
The cDNA is a strand of DNA complementary to a RNA molecule and made by the aid of an enzyme called reverse transcriptase. To make this happen, we need: 1 - buffer solution, to maintain the ideal pH; 2 - reverse transcriptase, which is the enzyme that will do the reverse transcription of RNA to cDNA ; 3 - dNTPs, the nucleotides used as substrate by the reverse transcriptase to synthesize cDNA; 4 - the RNA sample, that serves as a template for the reverse transcriptate; 5 - and primer to help the reaction start. We used anchored oligo dT primers.Those are a sequence of 18 to 25 Ts that bind to the poly A tail of messenger RNA (mRNA).
These reagents are present in the AppliedBiosystems' kit ''High Capacity cDNA Reverse Transcription Kit" and they are put together in ideal proportion, according to the manufacturer's protocol for cDNA synthesis, that occurs at 37 °C during 120 minutes.

So this is another step needed to achieve our goal. We hope you have enjoyed. Keep up with our blog !! Soon, we are going to write more about our adventures in the Lab.

segunda-feira, 6 de janeiro de 2014

The crew went to the circus

Today, Jan 6, we came back to work after a short holyday season break. Our two brave undergrad students deserved a couple of weeks off in order to recharge their batteries for the intense year to come.
They also deserved to celebrate the nice piece of work we have been doing during the first four months of this PEER Science project. For that reason, we went to the circus! More specifically we went to see Corteo, the Cirque du Soleil show that recently debut in Rio de Janeiro.

Corteo exhibit a dream of a circus clown. Not a regular dream but the dream of his own funeral! As it might be expected, the funeral of a clown is full of joy with traditional circus acts and surprising elements. Moreover, Corteo (as most of Cirque spectacles) shows that it is possible to “make magic” and delight the audience in limited space and with relatively simple tricks.
Since the first time I went to see Cirque du Soleil, I have been wondering the difference between the Cirque and any other common troupe. Yes, I believe the Cirque is common because it is made by normal people (although some appear not to have bones while others appear just to have muscle). The difference? Maybe the amount of money invested? Possibly! But if so, that is most probably not to acquire incredible expensive equipments but to hire the best people and to provide them an ever exciting environment. The most important difference relays on the people and not on the infrastructure! Just being passionate for what they do, they can devote the amount of time and energy in order to train for up to the perfection. Just unconditionally trusting on the work of a partner, one can throw yourself into the open air to be caught by the partner. Just being extremely fine tuned, artists and musicians can pace the rhythm for the 90 minutes of show. 

The take home message for my students: lets keep the passion for our work, lets keep the friendly and exciting environment of our group, lets work even harder and eventually we will became a kind of Cirque du Soleil in our field of science. In fact, as long as we travel through this road, it really does not matter how far we go. Trailing the path is more important than reaching its end.

Happy new year to all our followers.

quinta-feira, 24 de outubro de 2013

Nucleic acids extraction journey

The field collection is over but fun continues in the lab! Here, in the lab, is here we spend most of our time. After sampling fish tissue, the next step is to extract the total RNA content of each individual sample. In 6 days, our brave and hard working undergrad students, Paula and Maithê, have extracted RNA and the DNA from 88 fish livers.

Maithê (left) taking tubes out of the centrifuge with Paula observing at the back. Paula (right) dropping the supernadant out of the tubes to resuspend the RNA pellet.

The RNA will be used in our project. It is from these molecules that we will be able to sequence our favorite genes, CYP1A and AHR. The DNA will be sent to our collaborators at the National Museum of Rio de Janeiro for phylogenetics studies. At an appropriate moment, these two pieces of data will come together and eventually clarify some of the ecological roles of AHR/CYP1A in the evolution of loricariids.

RNA extraction is tricky! RNAses, enzymes that degrades RNA, are omnipresent! They are just every and anywhere; on your fingers, on your hair, on your saliva, on the dust (oww there are tons of that on the dust!), air etc! If you don’t take the necessary precautions, your sample will get contaminated, your RNA degraded and your time & patience lost. The thing is that you just get to know whether your RNA preparation is good or not at the very end of the process, when you run your sample in an agarose gel and actually see the RNA. Well truly you don’t see the RNA but instead the light emitted by a molecule, ethidium bromade, attached to it. The expected (or the good) result is to see two bands on the gel upon illuminating it with U.V. light. These two bands correspond to two of the ribosomal RNA molecules, the 18S and the 28S. Now, guess what did we get? No, I am not giving the answer! Check the photo of some of our extractions bellow and get to your own conclusions!

Great job girls! Very well done. Now, let’s keep the hard working and start cDNA preps, PCRs, cloning and sequencing. Well, that if the supplies arrives but this is story for another post.

quinta-feira, 10 de outubro de 2013

First field trip for fish sampling

The first field trip for fish sampling for our project was a great success, total of 88 fish of 20 different species of Siluriformes. The sampling occurred from Oct 2 - 7 through the north of Rio de Janeiro State to the south of Espirito Santo State and involved a total of 8 people: a senior professor (Prof. Paulo Buckup), a senior biologist (Décio), a PhD student (Carla Quijada), a master student (Emanuel Neuhaus), a former student and great collector (José), two drivers (Manuel and Lustosa) and myself (Thiago).

Few moments before de departure. Stand up: Paulo, Thiago, Carla, Décio and Emanuel. Sited: José 
After a long time away from the field, I honored my graduate degree, reviving the field collection excursions, entering the rivers, rolling stones (literally!), organizing cast nets etc.

Lots of species we are working with are really beautiful, like the one on my hand in the photo below, others are very tiny (2 cm long) making our work harder.

Meet the team

Currently our team is formed by the following people:

Dr. Thiago Estevam ParenteBrazilian PI: born in Rio de Janeiro, Thiago has been working with P450 enzymes since the age of 15 years old when took part of a science vocational program at FIOCRUZ. In 2006 obteined a bachelor degree in Biology major in Genetics from the UFRJ. In 2008 obteined his master degree in Cellular and Molecular Biology at FIOCRUZ.  In 2012 concluded his PhD in Biophysics at UFRJ. In 2005 Thiago was a summer fellow student at the WHOI, coming back in 2009 as a guest student.

Prof. Dr. Mauro de Freitas Rebelo - Brazilian co-PI: Mauro has a bachelor degree in Biology major in Marine Biology (UFRJ), a master degree in Biological Oceanography (FURG) and and a PhD in Biophysics (UFRJ). Mauro has a adjunct professor position at the UFRJ and is the head of the Environmental Molecular Biology Laboratory (BIOMA) that host our project. 

Dr. Mark HahnUS PI: Mark has a bachelor degree in Biological Sciences and a PhD in Toxicology. Mark is a senior scientist at the Biology Department of the WHOI where he has been working since 1992 and is currently the head of the Department and of the Hanh Lab.

Dr. John StegemanUS co-PI: John is a senior scientist at the Biology Department of the WHOI, the director of the Woods Hole Center for Oceans and Human Heath and the head of the Stegeman Lab. John has a bachelor degree in Biology and a PhD in Biochemistry. John has strong interest in Biochemical toxicology; metabolism and effects of pollutants and natural products; biochemistry, evolution and regulation of cytochrome P-450 isozymes; cloning, expression of P450s from diverse species; structure-function relationships involved in metabolism and effects of chemicals, as related to the susceptibility of different animal groups to the action of xenobiotics.

Dr. Adalberto Val - Adalberto is the director of the Brazilian National Institute for Amazon Research (INPA), the head of the ADAPTA project and of the Ecophysiology and Molecular Evolution Laboratory. He has a bachelor degree in Biology and a master and a PhD in Freshwater biology and fisheries. 

Dr. Prof. Paulo Buckup - Paulo has an adjunct professor position at the Rio de Janeiro National Museum, an academic and research center of UFRJ. Paulo has strong expertise in taxonomy, phylogeny and systematics of fishes. He has a bachelor degree in Biological Sciences, two master degrees, one in Biology and another in Biological Oceanography, and a PhD in Biological Sciences.

Paula Andrade - Paula is an highly motivated undergraduate student of Biology at the UFRJ.

Maithê Gaspar - Maithê is another highly motivated undergraduate student of Biology at the UFRJ.

Paula Calassara - Paula is our oldest undergraduate student. She is taking Biophysics at the UFRJ but is currently participating on the Science Without Borders internship in Pisa, Italy.

Our team list is incomplete. New people will be added soon.