Background: 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. Results: 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. Conclusion: 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.
Bibliographical noteFunding Information:
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number P50GM098911. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Wild isolates of C. remanei from Koffler Scientific Reserve at Jokers Hill, King City, Toronto, Ontario, CA were graciously provided by Asher Cutter's lab (University of Toronto). E. coli strain Rel606 DNA was provided by Brendan Bohannan's Lab (University of Oregon). Thanks to Paul D. Etter for his invaluable guidance and support in carrying out the molecular studies.
- De novo mutations
- Genetic heterogeneity
- Laboratory adaptation
- Minor alleles
- Next-generation sequencing
- PELE analysis