The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.
chip1_bp00x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 00x
annealing temperature = 45
PCR cycles = 40
chip1_bp01x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 01x
annealing temperature = 45
PCR cycles = 40
chip1_bp02x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 02x
annealing temperature = 45
PCR cycles = 40
chip1_bp05x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 05x
annealing temperature = 45
PCR cycles = 40
chip1_bp10x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 10x
annealing temperature = 45
PCR cycles = 40
chip1_bp20x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 20x
annealing temperature = 45
PCR cycles = 40
chip1_bp40x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 1
blocking primer concentration = 40x
annealing temperature = 45
PCR cycles = 40
chip2_bp00x_t40_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 40
PCR cycles = 40
chip2_bp00x_t45_c25
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 45
PCR cycles = 25
chip2_bp00x_t45_c30
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 45
PCR cycles = 30
chip2_bp00x_t45_c35
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 45
PCR cycles = 35
chip2_bp00x_t45_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 45
PCR cycles = 40
chip2_bp00x_t50_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 50
PCR cycles = 40
chip2_bp00x_t55_c40
Raw data in fastq format from the sequencing of the mock sample using a 314 Ion Torrent chip.
PCR conditions (see table 1 for details):
chip = 2
blocking primer concentration = 00x
annealing temperature = 55
PCR cycles = 40
Barcodes of the 11 species in the mock sample
Database with the COI barcodes of the eleven species obtained by Sanger sequencing (fasta file).
11_species.fasta
Barcodes of 188 Entomobryidae from Genbank
Database with the COI barcodes of 188 species of Entomobryidae (Collembola) obtained from Genbank (fasta file). Search: Entomobryidae[Organism] AND COI[Gene Name] AND Barcode[Keyword] (accessed on July 30th, 2014)
entomobryidae_20140730_coi_barcode.fasta