DOI: 10.5061/dryad.2p51n
title: Data from: Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods

description: 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.
description: chip1_bp00x_t45_c40Raw 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 = 40chip1_bp01x_t45_c40Raw 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 = 40chip1_bp02x_t45_c40Raw 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 = 40chip1_bp05x_t45_c40Raw 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 = 40chip1_bp10x_t45_c40Raw 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 = 40chip1_bp20x_t45_c40Raw 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 = 40chip1_bp40x_t45_c40Raw 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 = 40chip2_bp00x_t40_c40Raw 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 = 40chip2_bp00x_t45_c25Raw 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 = 25chip2_bp00x_t45_c30Raw 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 = 30chip2_bp00x_t45_c35Raw 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 = 35chip2_bp00x_t45_c40Raw 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 = 40chip2_bp00x_t50_c40Raw 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 = 40chip2_bp00x_t55_c40Raw 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 = 40Barcodes of the 11 species in the mock sampleDatabase with the COI barcodes of the eleven species obtained by Sanger sequencing (fasta file).11_species.fastaBarcodes of 188 Entomobryidae from GenbankDatabase 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