Extracting ‘legacy loci’ from an invertebrate sequence capture data set

Abstract

Sequence capture studies result in rich data sets comprising hundreds to thousands of targeted genomic regions that are superseding Sanger‐based data sets comprised of a few well‐known loci with historical uses in phylogenetics (‘legacy loci’). However, integrating sequence capture and Sanger‐based data sets is of interest as legacy loci can include different types of loci (e.g. mitochondrial and nuclear) across a potentially larger sample of species from past studies. Sequence capture data sets include nontargeted sequences, and there has been recent interest in extracting legacy loci from invertebrate data sets. Here, we use published legacy data from leaf‐footed bugs (Hemiptera: Coreoidea) to recover 15 mitochondrial and seven nuclear legacy loci from off‐target sequences in a sequence capture data set, explore approaches to improve legacy locus recovery, and combine these loci with sequence capture data for phylogenetic analysis. Two nuclear loci were determined to already be targeted by sequence capture baits. Most of the remaining loci were successfully recovered from off‐target sequences, but this recovery varied greatly. Additionally, complementing complete mitogenomes with additional reference mitochondrial sequences from a genetic depository did not offer improvement for most of our taxa; however, supplementing these reference sequences with extracted legacy loci offered ≥6% improvement across taxa for a given mitochondrial locus (negligible improvement for nuclear loci). Phylogenetic analysis of legacy and sequence capture data produced a topology generally congruent with recent studies, but support was lower. Thus, future studies may employ the approaches used in this study to integrate legacy data with newly generated sequence capture data sets without added expenses.

Low hybridization temperatures improve target capture success of invertebrate loci

“…This can be advantageous for phylogenomic studies that seek to generate more comprehensive datasets (taxon and character sampling) by integrating capture data with well-known markers of historical use in molecular phylogenetic studies. Several target capture studies, especially in vertebrates, have extracted legacy loci from off-target sequences in capture data (e.g., Meiklejohn et al, 2014; Amaral et al, 2015; Wang et al, 2017; Derkarabetian et al, 2019; Simon et al, 2019; Miller et al, 2022). Legacy locus data extracted for focal taxa can then be combined with loci retrieved from genetic depositories for other taxa of interest prior to phylogenetic analysis, as well as to assess possible errors and/or contamination.…”

“…Integrating legacy loci with target capture datasets has benefits, such as increasing the resolution power for phylogenetic inference and the inclusion of rare species with existing legacy data that are difficult to sample repeatedly (Branstetter et al, 2017; Derkarabetian et al, 2019; Zhang et al, 2019). While legacy locus data can be integrated with target capture data by designing baits from legacy loci (Branstetter et al, 2017; Simon et al, 2019; Hughes et al, 2021), this may increase the cost of custom probe kits because more baits may be required across more species due to higher substitution rates of some loci (e.g., mtDNA) and/or these baits may be included in a separate kit to prevent high copy number loci, like those on the mtDNA genome or the rRNA operon, from dominating capture data (Ströher et al, 2016; Pierce et al, 2017; Allio et al, 2020; Branstetter et al, 2021; Miller et al, 2022). Extracting legacy loci from off-target reads can circumvent some of these issues (Miller et al, 2022), and they have been successfully integrated with capture data despite their often-fewer numbers (compared to, e.g., 1000+ UCE loci) and/or introduction of large amounts of missing data (e.g., Simon et al, 2019; Miller et al, 2022).…”

“…As such, optimizing one or more in vitro target capture conditions may provide a more cost-effective solution to improve locus recovery. Studies in invertebrates typically employ a fixed hybridization temperature at 65°C as suggested by standard protocols (although temperatures are often not reported in AHE studies), with few studies having used lower temperatures during bait-target hybridization (Zhang et al, 2019;Braby et al, 2020;Emberts et al, 2020;Forthman et al, 2020;Miller et al;2022).…”

“…While legacy locus data can be integrated with target capture data by designing baits from legacy loci (Branstetter et al, 2017;Simon et al, 2019;Hughes et al, 2021), this may increase the cost of custom probe kits because more baits may be required across more species due to higher substitution rates of some loci (e.g., mtDNA) and/or these baits may be included in a separate kit to prevent high copy number loci, like those on the mtDNA genome or the rRNA operon, from dominating capture data (Ströher et al, 2016;Pierce et al, 2017;Allio et al, 2020;Branstetter et al, 2021;Miller et al, 2022). Extracting legacy loci from off-target reads can circumvent some of these issues (Miller et al, 2022), and they have been successfully integrated with capture data despite their often-fewer numbers (compared to, e.g., 1000+ UCE loci) and/or introduction of large amounts of missing data (e.g., Simon et al, 2019;Miller et al, 2022). Thus, having off-target reads may not always be detrimental in capture studies when designing legacy locus baits is less desirable, as long as targeted regions are also recovered.…”

“…However, off-targets reads may contain sequences from loci traditionally used in phylogenetic studies (herein referred to as “legacy loci”) (e.g., Amaral et al, 2015; Wang et al, 2017; Simon et al, 2019; Miller et al, 2022). Integrating legacy loci with target capture datasets has benefits, such as increasing the resolution power for phylogenetic inference and the inclusion of rare species with existing legacy data that are difficult to sample repeatedly (Branstetter et al, 2017; Derkarabetian et al, 2019; Zhang et al, 2019).…”