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

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

SUBMITTER: Marmesat E 

PROVIDER: S-EPMC4905633 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature