New targets acquired: improving locus recovery from the Angiosperms353 probe set

Abstract

Universal target enrichment kits maximise utility across wide evolutionary breadth while minimising the number of baits required to create a cost-efficient kit. Locus assembly requires a target reference, but the taxonomic breadth of the kit means that target references files can be phylogenetically sparse. The Angiosperms353 kit has been successfully used to capture loci throughout angiosperms but includes sequence information from 6−18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, reducing locus recovery. We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a mega353 target file, with each gene represented by 17−373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene datasets. Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 31%, increased loci recovery at 75% length by 61.9%, and increased the total length of the concatenated loci by 30%. The mega353 file and associated scripts are available at: https://github.com/chrisjackson−pellicle/NewTargets

On the potential of Angiosperms353 for population genomic studies

“…Although much of the DNA extracted from the herbarium specimens was degraded, a third of the samples required further fragmentation before library preparation to increase the number of fragments in our desired size range (~500 bp P = 0.292). The use of the "mega353" target file (McLay et al, 2021) substantially increased the gene recovery for many samples: more than 50 additional genes were recovered at 50% target length for Morus celtidifolia Kunth, Sporobolus cryptandrus (Torr.) A.…”

“…With newer sequencing technologies including the Illumina NovaSeq SP, which has an output of 400,000,000 reads per lane, our results suggest that massive pooling of samples (at least 1500, given flexible library indices) could be achieved. We also saw a marked increase in gene recovery when using the "mega353" (McLay et al, 2021) target file in HybPiper. This target file was constructed using a larger curated set of orthologs for each Angiosperms353 probe set, compared with a target file constructed only from the representative sequences used to design the Angiosperms353 probes (Johnson et al, 2019).…”

“…Angiosperms353 genes were recovered from cleaned Illumina reads using HybPiper (Johnson et al, 2016a). We used the "mega353" target file (McLay et al, 2021) to recover sequences; this file contains substantially more representative sequences per gene than the original Angiosperms353 target file. Briefly, the HybPiper workflow identifies potential matches between reads and target genes (using BLASTX [Camacho et al, 2009]) to allow for high phylogenetic distance between targeted sequences and sample reads, sorts the reads by gene, and conducts de novo assembly separately on each gene, mitigating the need for a reference sequence for non-model organisms.…”

Target sequence capture in orchids: Developing a kit to sequence hundreds of single‐copy loci

“…The lower number of genes recovered from the Cypripedioideae and Orchidoideae clades and the 137 genes with low capture rates across the Orchidaceae can potentially be addressed with future reference-genome sequences for these subfamilies and improvements to target capture bioinformatic pipelines. For example, capture rates can be improved by using subfamilyspecific exon files for read mapping or clustering methods to select multiple sequences per loci representative of sequence diversity across the family (Johnson et al, 2019;McLay et al, 2021). Previous analyses of tribal relationships within the Epidendroideae subfamily have found a polytomy among the Cymbideae, Vandeae, and Epidendreae tribes (Freudenstein et al, 2004;Neubig et al, 2009).…”

Relative performance of customized and universal probe sets in target enrichment: A case study in subtribe Malinae

“…Although we did not detect a great improvement in the proportion of mapped reads when optimizing the Angiosperms353 reference using the Malus genome, we saw an increase in locus recovery: the percentage of loci with both a ≥75% target length and accessions presence was 53% for Angiosperms353 vs. 73% for the Malinae-optimized reference. Bioinformatically altering the performance of Angiosperms353 could alternatively be achieved by increasing the number of taxa per locus in the reference file used for HybPiper analysis (McLay et al, 2021). Alternatively, the optimization of Angiosperms353 could be performed during probe design by replacing the Angiosperms353 sequences with appropriate orthologs from a genomic resource of the study group, such as genome skimming data (Jantzen et al, 2020), although the authors of the study emphasize that this genomic resource should be closely related to the study group.…”