High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).
Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing.



We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation.



We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. jpreston@molbio.uoregon.edu.
Jessica L
JL
Preston
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.
Ariel E
AE
Royall
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.
Melissa A
MA
Randel
Institute of Ecology and Evolution, University of Oregon, Eugene, Oregon, USA.
Kristin L
KL
Sikkink
Institute of Ecology and Evolution, University of Oregon, Eugene, Oregon, USA.
Patrick C
PC
Phillips
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.
Eric A
EA
Johnson
D000483:Alleles; D004926:Escherichia coli / Q000235:genetics; D005787:Gene Frequency; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards
De novo mutations; Genetic heterogeneity; Laboratory adaptation; Minor alleles; Next-generation sequencing; PELE analysis; SNPs