Heavy parallel sequencing of PCR amplicons.
We here suggest an approach to large scale parallel sequencing where many samples (in theory, up to several thousands) can be sequenced at the same time. The methodology is designed for targeting specific genes, and depends on an initial polymerase chain reaction (PCR) where tagged primers are used.
Each sample (e.g., each individual) has its specific tag, which functions as an identification of the sample. Due to the sample-specific tag, otherwise unrelated samples might be mixed into the same sequencing run.
To show the applicability of the methodology, we here apply the method to hemoglobins in cod populations from the Faroe Bank and the Faroe Plateau. In addition to sequencing genomic DNA, we will also investigate the expression of hemoglobins at the levels of mRNA (by making cDNA that subsequently is sequenced by the same technique as described above) and protein (by mass spectrometry).
The results will help to understand the structures of the Faroese cod population and its subpopulations. They will also indicate whether there are regulatory mechanisms for hemoglobins both at transcriptional and translational levels. Furthermore, hemoglobin allele expression has previously been correlated to both growth rate and water temperatures. Therefore the results may have wide implications, for example in studies of the unusually fast growth rate of cod from the Faroe Bank, or in studies of the impact of climate change on cod populations.
It should further be noted that the described method can be used in any organism, it be cod, bacteria or humans, including large scale sequencing of human genes involved in heritable diseases.
Svein-Ole Mikalsen, Ph.D.