title: megasat: automated inference of microsatellite genotypes from sequence data.
abstract: megasat is software that enables genotyping of microsatellite loci using next-generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. megasat reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important feature of megasat is the generation of histograms of the length-frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. megasat is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demonstrate megasat using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0-1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs.
authors.affiliation: Faculty of Computer Science, Dalhousie University, 6050 University Avenue, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Luyao
authors.initials: L
authors.lastname: Zhan
authors.affiliation: Marine Gene Probe Laboratory, Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Paterson
authors.affiliation: Evolution Behaviour and Environment Group, University of Sussex, Sussex House, Falmer, Brighton, BN1 9RH, UK.
authors.forename: Bonnie A
authors.initials: BA
authors.lastname: Fraser
authors.affiliation: Marine Gene Probe Laboratory, Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Beth
authors.initials: B
authors.lastname: Watson
authors.affiliation: Salmonids Section, Science Branch, Department of Fisheries and Oceans Canada, 80 East White Hills Road, St. John's, Newfoundland, A1C 5X1, Canada.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Bradbury
authors.affiliation: Faculty of Computer Science, Dalhousie University, 6050 University Avenue, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Praveen
authors.initials: P
authors.lastname: Nadukkalam Ravindran
authors.affiliation: Department of Biology, University of California, Riverside, CA, 92521, USA.
authors.forename: David
authors.initials: D
authors.lastname: Reznick
authors.affiliation: Faculty of Computer Science, Dalhousie University, 6050 University Avenue, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Beiko
authors.affiliation: Marine Gene Probe Laboratory, Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Bentzen
mesh_terms: D000818:Animals; D019295:Computational Biology / Q000379:methods*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D018895:Microsatellite Repeats; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011036:Poecilia / Q000145:classification / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
keywords: animal mating/breeding systems; bioinformatics/phyloinformatics; captive populations; conservation genetics; landscape genetics; population genetics - empirical