Introduction: Amplicon deep sequencing of environmental samples is one of the promising method in describing biodiversity structure of a given environment. However this approach the emphasis is not given to the quantitative result although it was already shown in ecological studies that the description of biodiversity need to include species abundance and not simply presence or absence (1). In fact, one of the critical steps in environmental sequencing is the choice of suitable primer set yielding the highest sequence diversity nearly to the real one both in quantity and quality diversity. Amplicon diversity screening depends upon amplification bias rate that universal primers can have during amplification of bulk DNA. The main aim of this study was the evaluation of amplification bias level of universal primers along all sequencing protocol preparation steps of our environmental samples. Folmer primer set (2) was selected to amplify the DNA barcode region (COI) known by its capacity to identify a broad spectrum animal taxa (3). The study was conducted on two naturally collected samples (MPE4, MPE5) from the same locality and one artificial. The first two samples were the yield of McPhail traps placed in litter soil layer of chestnut forest while the third consists on equal DNA concentration pool of four species extracted from the above natural samples. In addition, species of Carabidae family within MPE4 and MPE5 were identified morphologically and the barcode region of these species was obtained by sanger sequencing. The organism biomass within MPE4 was equalized between carabids while that of MPE5 was left intact. Methods: To evaluate amplification bias of Folmer primer set within naturally complex sample, specific Real-time PCR primers were designed on single individual COI sequences to monitor the abundance ratio of the organisms under study after amplification with universal primers. MPE4 and MPE5 were amplified using Folmer primers and submitted to 454 pyrosequencing using shotgun library preparation and passing by genomic amplification step with phi29 polymerase. Primer specificity of two species (CC1 and LL1) out of four was tested in qPCR on both single individuals and on pooled DNA before and after amplification with universal primers. The abundance of the CC1 and LL1 was monitored in qPCR over all sequencing steps: Bulk extracted DNA, Folmer amplicons, phi29 products and nebulized products. In the case of artificial sample, these two species were quantified just for in artificial pool DNA and after amplification with Folmer primers. Quantification with qPCR was performed using the MAK2 algorithm (4) implemented in a R routine. Finally, ratio intensity of the two species DNA in all experimental steps was compared to their biomass ratio and species assigned read abundance obtained after signal de-noising with Ampliconoise suite(5). Results: Regarding the artificial sample where DNA concentrations were equal across species, Folmer primers showed a significant bias toward CC1 over LL1. In MPE4, where species biomass was equalized, the bulk mtDNA signal ratio was already biased towards CC1 (qPCR signal intensity of CC1/LL1 = 53.92), and PCR amplification with universalprimers increased substantially that bias (CC1/LL1 =163.76). This ratio decreased in phi29 amplification, nebulization, and sequencing steps to reach the value of 1.55 in terms of sequence abundance that is near to the biomass ratio (Fig.1). MPE5 sample followed the same qualitative profile of the MPE4 with preference for CC1 in DNA extraction and amplification steps and re-equalization towards LL1 in the following steps (Fig.2). In conclusion, Folmer primer set showed a significant amplification bias in PCR reactions when used in both complex and simple bulk DNA. Sequence abundance seem to be affected by the activity of Phi29 polymerases and nebulization during sequencing library preparation and their effect seems towards an equalization of the templates.
Exploring Folmer Universal Primers Bias In Environmental 454 Pyrosequencing Using Real-Time PCR Approach
Bachir Balech;Saverio Vicario
2011
Abstract
Introduction: Amplicon deep sequencing of environmental samples is one of the promising method in describing biodiversity structure of a given environment. However this approach the emphasis is not given to the quantitative result although it was already shown in ecological studies that the description of biodiversity need to include species abundance and not simply presence or absence (1). In fact, one of the critical steps in environmental sequencing is the choice of suitable primer set yielding the highest sequence diversity nearly to the real one both in quantity and quality diversity. Amplicon diversity screening depends upon amplification bias rate that universal primers can have during amplification of bulk DNA. The main aim of this study was the evaluation of amplification bias level of universal primers along all sequencing protocol preparation steps of our environmental samples. Folmer primer set (2) was selected to amplify the DNA barcode region (COI) known by its capacity to identify a broad spectrum animal taxa (3). The study was conducted on two naturally collected samples (MPE4, MPE5) from the same locality and one artificial. The first two samples were the yield of McPhail traps placed in litter soil layer of chestnut forest while the third consists on equal DNA concentration pool of four species extracted from the above natural samples. In addition, species of Carabidae family within MPE4 and MPE5 were identified morphologically and the barcode region of these species was obtained by sanger sequencing. The organism biomass within MPE4 was equalized between carabids while that of MPE5 was left intact. Methods: To evaluate amplification bias of Folmer primer set within naturally complex sample, specific Real-time PCR primers were designed on single individual COI sequences to monitor the abundance ratio of the organisms under study after amplification with universal primers. MPE4 and MPE5 were amplified using Folmer primers and submitted to 454 pyrosequencing using shotgun library preparation and passing by genomic amplification step with phi29 polymerase. Primer specificity of two species (CC1 and LL1) out of four was tested in qPCR on both single individuals and on pooled DNA before and after amplification with universal primers. The abundance of the CC1 and LL1 was monitored in qPCR over all sequencing steps: Bulk extracted DNA, Folmer amplicons, phi29 products and nebulized products. In the case of artificial sample, these two species were quantified just for in artificial pool DNA and after amplification with Folmer primers. Quantification with qPCR was performed using the MAK2 algorithm (4) implemented in a R routine. Finally, ratio intensity of the two species DNA in all experimental steps was compared to their biomass ratio and species assigned read abundance obtained after signal de-noising with Ampliconoise suite(5). Results: Regarding the artificial sample where DNA concentrations were equal across species, Folmer primers showed a significant bias toward CC1 over LL1. In MPE4, where species biomass was equalized, the bulk mtDNA signal ratio was already biased towards CC1 (qPCR signal intensity of CC1/LL1 = 53.92), and PCR amplification with universalprimers increased substantially that bias (CC1/LL1 =163.76). This ratio decreased in phi29 amplification, nebulization, and sequencing steps to reach the value of 1.55 in terms of sequence abundance that is near to the biomass ratio (Fig.1). MPE5 sample followed the same qualitative profile of the MPE4 with preference for CC1 in DNA extraction and amplification steps and re-equalization towards LL1 in the following steps (Fig.2). In conclusion, Folmer primer set showed a significant amplification bias in PCR reactions when used in both complex and simple bulk DNA. Sequence abundance seem to be affected by the activity of Phi29 polymerases and nebulization during sequencing library preparation and their effect seems towards an equalization of the templates.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
