PCR Strategies for Complete Allele Calling in Multigene Families Using High-Throughput Sequencing Approaches

PLoS One. 2016 Jun 13;11(6):e0157402. doi: 10.1371/journal.pone.0157402. eCollection 2016.

Abstract

The characterization of multigene families with high copy number variation is often approached through PCR amplification with highly degenerate primers to account for all expected variants flanking the region of interest. Such an approach often introduces PCR biases that result in an unbalanced representation of targets in high-throughput sequencing libraries that eventually results in incomplete detection of the targeted alleles. Here we confirm this result and propose two different amplification strategies to alleviate this problem. The first strategy (called pooled-PCRs) targets different subsets of alleles in multiple independent PCRs using different moderately degenerate primer pairs, whereas the second approach (called pooled-primers) uses a custom-made pool of non-degenerate primers in a single PCR. We compare their performance to the common use of a single PCR with highly degenerate primers using the MHC class I of the Iberian lynx as a model. We found both novel approaches to work similarly well and better than the conventional approach. They significantly scored more alleles per individual (11.33 ± 1.38 and 11.72 ± 0.89 vs 7.94 ± 1.95), yielded more complete allelic profiles (96.28 ± 8.46 and 99.50 ± 2.12 vs 63.76 ± 15.43), and revealed more alleles at a population level (13 vs 12). Finally, we could link each allele's amplification efficiency with the primer-mismatches in its flanking sequences and show that ultra-deep coverage offered by high-throughput technologies does not fully compensate for such biases, especially as real alleles may reach lower coverage than artefacts. Adopting either of the proposed amplification methods provides the opportunity to attain more complete allelic profiles at lower coverages, improving confidence over the downstream analyses and subsequent applications.

MeSH terms

  • Alleles
  • Animals
  • Base Sequence
  • DNA / genetics
  • DNA Copy Number Variations
  • DNA Primers / genetics
  • Exons
  • Genes, MHC Class I*
  • High-Throughput Nucleotide Sequencing / methods*
  • Lynx / genetics*
  • Multigene Family*
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA / methods*

Substances

  • DNA Primers
  • DNA

Grants and funding

Funding for this project was provided by the Dirección General de Investigación Cinetífica y Técnica, Spanish Science and Innovation Ministry, through grants CGL2010-21540/BOS and CGL2013-47755-P. Elena Marmesat received a JAE predoctoral grant from CSIC (Spanish National Research Council) and a short stay grant within the framework of the ESF activity on 'Conservation Genomics: Amalgamation of Conservation Genetics and Ecological and Evolutionary Genomics'. The authors acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.