Microhaplotypes provide increased power from short‐read DNA sequences for relationship inference

Abstract

The accelerating rate at which DNA sequence data are now generated by high-throughput sequencing instruments provides both opportunities and challenges for population genetic and ecological investigations of animals and plants. We show here how the common practice of calling genotypes from a single SNP per sequenced region ignores substantial additional information in the phased short-read sequences that are provided by these sequencing instruments. We target sequenced regions with multiple SNPs in kelp rockfish (Sebastes atrovirens) to determine "microhaplotypes" and then call these microhaplotypes as alleles at each locus. We then demonstrate how these multi-allelic marker data from such loci dramatically increase power for relationship inference. The microhaplotype approach decreases false-positive rates by several orders of magnitude, relative to calling bi-allelic SNPs, for two challenging analytical procedures, full-sibling and single parent-offspring pair identification. We also show how the identification of half-sibling pairs requires so much data that physical linkage becomes a consideration, and that most published studies that attempt to do so are dramatically underpowered. The advent of phased short-read DNA sequence data, in conjunction with emerging analytical tools for their analysis, promises to improve efficiency by reducing the number of loci necessary for a particular level of statistical confidence, thereby lowering the cost of data collection and reducing the degree of physical linkage amongst markers used for relationship estimation. Such advances will facilitate collaborative research and management for migratory and other widespread species.

Dispersal of a nearshore marine fish connects marine reserves and adjacent fished areas along an open coast

“…Genetic results indicating high levels of self‐retention should always be coupled with a complete explanation of the methods used to generate those results (Harrison, Saenz‐Agudelo, Planes, Jones, & Berumen, 2013a, 2013b; Saenz‐Agudelo, Jones, Thorrold, & Planes, ), because the ability to accurately identify true relationships hinges on the statistical power of a given panel of genetic markers, and uncertainty involved in genetic and parentage analyses can lead to uncertainty in estimates of dispersal (Kaplan et al, ). Note that it is exceedingly difficult to discriminate full and half siblings, as well as other categories of related individuals, with genetic data (Baetscher et al, ). We used a conservative set of criteria for retention of siblings, and thus probably excluded some true sibling pairs from the final set.…”

“…Each microhaplotype locus is 100–130 bp and contains at least one SNP. Genotyping followed the Genotyping‐in‐Thousands by sequencing (GT‐seq) protocol of Campbell, Harmon, and Narum (), with modifications as described by Baetscher et al (). Each sequencing library consisted of 384 individually indexed samples.…”

“…The read depth ratio is calculated as the number of reads for each haplotype divided by the number of reads of the most common haplotype in that individual at that locus. This read depth ratio criterion is intended to remove spurious haplotypes caused by index switching and genotyping error, which may not be identified by the read depth threshold alone (Baetscher et al, ). Additionally, the presence of more than two haplotypes in an individual at multiple loci after filtering provided an opportunity to identify potentially contaminated DNA extracts.…”

“…While the identification of parent–offspring relationships has been the dominant form of relationship inference used in the study of dispersal in the marine realm, Schunter, Pascual, Garza, Raventos, and Macpherson () showed how combining identification of sibling groups with parentage analysis provides a more complete description of dispersal patterns. Such sibship analysis has since seen additional use in the study of marine fishes (e.g., Ottmann et al, ), but Baetscher, Clemento, Ng, Anderson, and Garza () have shown that most such analyses have lacked sufficient power to accurately identify sibling groups.…”

A versatile Rapture (RAD‐Capture) platform for genotyping marine turtles

“…For example, researchers interested in basic population structure and individual assignment may wish to design kits with a subset of only several hundred informative targets, increasing the per locus depth of coverage in each sample (Hoffberg et al, ; Mastretta‐Yanes et al, ). Researchers can also target regions with multiple SNPs to develop multiallelic microhaplotype markers that provide greater power per locus (McKinney, Seeb, & Seeb, ), and can be particularly useful for applications such as kinship studies (Baetscher, Clemento, Ng, Anderson, & Garza, ). Finally, the ability to repeatedly capture the same genomic regions facilitates studies conducted over broader time periods (e.g., examining trends across many nesting seasons or even generations) or spatial scales (e.g., collaborating laboratories can generate and share data between foraging and nesting grounds).…”