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Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results.

Brandariz-Fontes C, Camacho-Sanchez M, Vilà C, Vega-Pla JL, Rico C, Leonard JA - Sci Rep (2015)

Bottom Line: We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps.Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads.Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles.

View Article: PubMed Central - PubMed

Affiliation: 1] Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain [2] Facultad de Medicina Veterinaria, Universidad de Panamá, Panama.

ABSTRACT
Library preparation protocols for high-throughput DNA sequencing (HTS) include amplification steps in which errors can build up. In order to have confidence in the sequencing data, it is important to understand the effects of different Taq polymerases and PCR amplification protocols on the DNA molecules sequenced. We compared thirteen enzymes in three different marker systems: simple, single copy nuclear gene and complex multi-gene family. We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps. We find that enzyme choice has a large impact on the proportion of correct sequences recovered. The most complex marker systems yielded fewer correct reads, and the proportion of correct reads was greatly affected by the enzyme used. Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads. Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles. Consequently, enzyme selection for downstream HTS has important consequences, especially in complex genetic systems.

No MeSH data available.


Related in: MedlinePlus

Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares; medium: two alleles, circles; and complex: multiple alleles, triangles) using standard PCR conditions (open) and modified PCR conditions to reduce chimera formation (gray).The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1.
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f1: Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares; medium: two alleles, circles; and complex: multiple alleles, triangles) using standard PCR conditions (open) and modified PCR conditions to reduce chimera formation (gray).The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1.

Mentions: There was a significant effect of the enzyme on the quality of the sequences obtained (proportion of reads with a correct sequence) for all tests (p < 0.001 in all cases). In general, Biotaq produced the lowest portion of correct reads across all tests whereas Phusion, Pwo and KapHF worked best (Supplementary File 1). For Test 1 (with only one allele expected per individual), all the enzymes that successfully amplified DNA (11 out of 13) yielded from 50–53% (OneTaq and Biotaq) to 88–92% (Phusion, Pwo and KapHF) correct reads (Figure 1). For Test 2, the proportion of correct reads was on average 23% lower than for Test 1. There was also more variation between the enzymes, with correct reads ranging from 2% (Biotaq) to 84% (Phusion) (Figure 1). For Test 3, the multigene family marker system, the recovery of correct sequences ranged from 17–20% (Biotaq, HotStar and Roche Taq) to 65–71% (Phusion and FastStart) (Figure 1).


Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results.

Brandariz-Fontes C, Camacho-Sanchez M, Vilà C, Vega-Pla JL, Rico C, Leonard JA - Sci Rep (2015)

Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares; medium: two alleles, circles; and complex: multiple alleles, triangles) using standard PCR conditions (open) and modified PCR conditions to reduce chimera formation (gray).The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306961&req=5

f1: Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares; medium: two alleles, circles; and complex: multiple alleles, triangles) using standard PCR conditions (open) and modified PCR conditions to reduce chimera formation (gray).The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1.
Mentions: There was a significant effect of the enzyme on the quality of the sequences obtained (proportion of reads with a correct sequence) for all tests (p < 0.001 in all cases). In general, Biotaq produced the lowest portion of correct reads across all tests whereas Phusion, Pwo and KapHF worked best (Supplementary File 1). For Test 1 (with only one allele expected per individual), all the enzymes that successfully amplified DNA (11 out of 13) yielded from 50–53% (OneTaq and Biotaq) to 88–92% (Phusion, Pwo and KapHF) correct reads (Figure 1). For Test 2, the proportion of correct reads was on average 23% lower than for Test 1. There was also more variation between the enzymes, with correct reads ranging from 2% (Biotaq) to 84% (Phusion) (Figure 1). For Test 3, the multigene family marker system, the recovery of correct sequences ranged from 17–20% (Biotaq, HotStar and Roche Taq) to 65–71% (Phusion and FastStart) (Figure 1).

Bottom Line: We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps.Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads.Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles.

View Article: PubMed Central - PubMed

Affiliation: 1] Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain [2] Facultad de Medicina Veterinaria, Universidad de Panamá, Panama.

ABSTRACT
Library preparation protocols for high-throughput DNA sequencing (HTS) include amplification steps in which errors can build up. In order to have confidence in the sequencing data, it is important to understand the effects of different Taq polymerases and PCR amplification protocols on the DNA molecules sequenced. We compared thirteen enzymes in three different marker systems: simple, single copy nuclear gene and complex multi-gene family. We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps. We find that enzyme choice has a large impact on the proportion of correct sequences recovered. The most complex marker systems yielded fewer correct reads, and the proportion of correct reads was greatly affected by the enzyme used. Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads. Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles. Consequently, enzyme selection for downstream HTS has important consequences, especially in complex genetic systems.

No MeSH data available.


Related in: MedlinePlus