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!

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