Technical Comment on ¿Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses¿
Zsuzsanna Guba, Hungarian Natural History Museum
12 September 2012
Technical Comment on ¿Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses¿
Tamas Zeke , Zsuzsanna Guba
Laboratory of Molecular Anthropology, Hungarian Natural History Museum, Budapest, Hungary. Using pooled samples in molecular biology is a widely known/used approach. It can be very fruitful to collect sequence data from a large scale collection of samples. However, it is to be used only in case of individual samples which can be identified throughout the whole analysis. In case of ancient pollen samples, it is not wise to use pooled samples in experiments, because from sequence similarity alone you can not track back the identity of an individual. This is similar to molecular cloning where the identity of the individual in the experimental reproduction can only be assured by separation of the single DNA molecules. For extant pollen samples, geographical patterns of haplogroups from pine species can confidently be determined by the support of the accurate molecular cloning techniques. As a result, with well defined sequence information in hands, haplotype lineages of mediterranean Abies species with their population distribution and genetic contact history were possible to be determined in details (Liepelt et al., 2010).
It is also widely known that it is not only the low number of template molecules that means a real challenge in ancient DNA studies. Detecting possible template heterogenity in PCR is hard, but required for getting autochtonous aDNA sequences and this is always stressed in ancient human DNA experiments. In the mixture of 5-6 pollen grains, one just produces the phenomenon which is to be avoided: the very likely presence of heterogenous template molecules in a PCR tube. In solving of the detection of template heterogenity, cloning is usually adviced and expected, supplemented with numerous replication of PCR and sequencing reactions (Cooper and Poinar, 2000). Reamplification can not exclude the template heterogenity at all, as it is known that some ¿better¿ templates can be favoured by polymerase enzyme in PCR.
¿Cloning is generally performed in the case of aDNA studies to detect mosaic sequences as a result of jumping PCR between sequences, to identify amplification errors due to post-mortem DNA damage in the template and to detect PCR-slippage in the case of microsatellites¿ This statement taken from Magyari et al. (2011) appears a very artificial one in the key part of the Materials and Methods, and does not seem for readers a real part of the experimental design. Moreover, no real explanation appears in other part of the publication. In fact, after the method of mixed pollen sampling, the above statement is meaningless, because several sources of mosaic sequences are provided and the cloning procedure itself is not suitable to distinguish among them. That is why a clearer approach would be required for the experimental background. They also stated clearly in the article that the mixing of several (5-6) pollen grains can theoretically cause the amplification of more than one cpDNA sequence with different base order. Mosaic sequences as a result of jumping PCR between sequences would be also possible as a PCR modification. However, the mixing of several pollens in one extraction is not likely to cause jumping PCR. This is an invalid statement! The main reason of this PCR error is not the presence of sequence mixtures, but base modifications, which can occur in homogenous extraction as well. But, again, it is a source for extra sequence heterogeneity in sample mixtures. Furthermore, as stated: ¿Importantly, generation of mosaic sequences was excluded in our case due to the uniparental inheritance of the chloroplast genome in spruce¿. Once a pollen mixture is applied in a single extraction, the previous statement is irrelevant as well as misleading, because ¿generation¿ means something else, ¿generating¿ should have been written here!
Technical Comment on ¿Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses¿
12 September 2012
Technical Comment on ¿Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses¿
Tamas Zeke , Zsuzsanna Guba
Laboratory of Molecular Anthropology, Hungarian Natural History Museum, Budapest, Hungary. Using pooled samples in molecular biology is a widely known/used approach. It can be very fruitful to collect sequence data from a large scale collection of samples. However, it is to be used only in case of individual samples which can be identified throughout the whole analysis. In case of ancient pollen samples, it is not wise to use pooled samples in experiments, because from sequence similarity alone you can not track back the identity of an individual. This is similar to molecular cloning where the identity of the individual in the experimental reproduction can only be assured by separation of the single DNA molecules. For extant pollen samples, geographical patterns of haplogroups from pine species can confidently be determined by the support of the accurate molecular cloning techniques. As a result, with well defined sequence information in hands, haplotype lineages of mediterranean Abies species with their population distribution and genetic contact history were possible to be determined in details (Liepelt et al., 2010).
It is also widely known that it is not only the low number of template molecules that means a real challenge in ancient DNA studies. Detecting possible template heterogenity in PCR is hard, but required for getting autochtonous aDNA sequences and this is always stressed in ancient human DNA experiments. In the mixture of 5-6 pollen grains, one just produces the phenomenon which is to be avoided: the very likely presence of heterogenous template molecules in a PCR tube. In solving of the detection of template heterogenity, cloning is usually adviced and expected, supplemented with numerous replication of PCR and sequencing reactions (Cooper and Poinar, 2000). Reamplification can not exclude the template heterogenity at all, as it is known that some ¿better¿ templates can be favoured by polymerase enzyme in PCR.
¿Cloning is generally performed in the case of aDNA studies to detect mosaic sequences as a result of jumping PCR between sequences, to identify amplification errors due to post-mortem DNA damage in the template and to detect PCR-slippage in the case of microsatellites¿ This statement taken from Magyari et al. (2011) appears a very artificial one in the key part of the Materials and Methods, and does not seem for readers a real part of the experimental design. Moreover, no real explanation appears in other part of the publication. In fact, after the method of mixed pollen sampling, the above statement is meaningless, because several sources of mosaic sequences are provided and the cloning procedure itself is not suitable to distinguish among them. That is why a clearer approach would be required for the experimental background. They also stated clearly in the article that the mixing of several (5-6) pollen grains can theoretically cause the amplification of more than one cpDNA sequence with different base order. Mosaic sequences as a result of jumping PCR between sequences would be also possible as a PCR modification. However, the mixing of several pollens in one extraction is not likely to cause jumping PCR. This is an invalid statement! The main reason of this PCR error is not the presence of sequence mixtures, but base modifications, which can occur in homogenous extraction as well. But, again, it is a source for extra sequence heterogeneity in sample mixtures. Furthermore, as stated: ¿Importantly, generation of mosaic sequences was excluded in our case due to the uniparental inheritance of the chloroplast genome in spruce¿. Once a pollen mixture is applied in a single extraction, the previous statement is irrelevant as well as misleading, because ¿generation¿ means something else, ¿generating¿ should have been written here!
Competing interests
None declared