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.