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The construction of a cDNA library involves making a DNA 'copy' of the mRNA because the aim is to synthesise a large set of recombinant plasmid molecules, each containing a different cDNA and each propagated in E. coli. The heyday of cDNA library construction predates the invention of PCR. The key difference between the two approaches that you are ...


2

When you say RPKM do you mean crude RPKM or the estimates that you get using expectation maximization methods like cufflinks and eXpress? It is better if you get your RPKM or FPKM values from one of these programs because you can differentiate between transcript variants. I have mostly used cufflinks and eXpress. Cufflinks package is better for multiple ...


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The Next-Gen sequencers cannot sequence a very long stretch of DNA with good reliability (~150 for the recent model- HiSeq2000; even less for older models such as GA (40), GA-II (70), GA-IIx (90)). For increasing the confidence in a certain hit, it was sequenced from both the ends. For example, if you have selected 500bp DNA fragment, then after ligating ...


4

In Illumina sequencing, the DNA is (usually randomly) sheared into fragments. For paired end sequencing, fragments of a specific size range are selected and then sequenced from both sides. This results in two reads for each fragment. As read length is fixed, also the remaining "middle part" of the fragment is in a specific size range. In some cases there is ...



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