The Effects of Herbarium Specimen Characteristics on Short-Read NGS Sequencing Success in Nearly 8000 Specimens: Old, Degraded Samples Have Lower DNA Yields but Consistent Sequencing Success

Abstract

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies.

Global Phylogeny of the Brassicaceae Provides Important Insights into Gene Discordance

“…This method offers several important advantages over full genome sequencing and other modern sequencing methods, notably a combination of broad taxonomic application with high efficiency on non-model species, broad phylogenetic informativeness at both deep and shallow nodes, and cost-efficiency (Lemmon and Lemmon 2013). Because the method relies on short DNA sequences (200-400 bp), may work with very low DNA starting amount (down to 5 ng), and is rather insensitive to DNA degradation, it is also ideally suited to work with (up to centuries-old) material from natural history museums, allowing access to rare and previously ‘locked’ specimens (Särkinen et al 2012; Hart et al 2016; Brewer et al 2019; Forrest et al 2019; Alsos et al 2020; Bakker et al 2020; Folk et al 2021; Kates et al 2021). In addition, target capture sequencing of leaves generates a considerable sequence bycatch in the form of off-target reads (‘genome skimming’) that represent a wealth of chloroplast genes, allowing simultaneous reconstruction of the plastome (Weitemier et al 2014).…”

Ancient DNA extraction methods for herbarium specimens: When is it worth the effort?

“…One would expect that plants collected and desiccated in cool and dry environments would yield higher quantities of less‐degraded DNA than plants collected under wet and tropical conditions. Although thus far only a few studies have tried to investigate these aspects (e.g., Kates et al, 2021 ), Bakker et al ( 2016 ) found that, based on read assembly results, the fragmentation effects caused by the age of the sample were more consistent in materials from wet and tropical environments, probably due to the longer and more destructive preparation methods used (e.g., heat, alcohol).…”

“…These proportions are comparable to those obtained using the same kit with fresh (silica gel–dried) samples (data not published). The target enrichment has already been successfully applied to relatively old herbarium specimens (Hart et al, 2016 ; Villaverde et al, 2018 ; Kates et al, 2021 ); however, for very old specimens (>200 years), methods based on genome skimming and the assembly of multicopy genome regions (e.g., organellar DNA), coupled with single‐stranded DNA library preparation, perform better than the target enrichment of single‐copy nuclear regions (Bakker, 2017 ). Our results confirm the potential of the latter technique, even when applied to herbarium specimens up to 200 years old.…”

Fagalean phylogeny in a nutshell: Chronicling the diversification history of Fagales

“…The DNA sequence data used here were extracted from the set of 100 low-copy nuclear loci used in a project (Folk et al 2021a; Kates et al 2021) that sampled ca. 15,000 species of the nitrogen-fixing clade of angiosperms, of which Fagales are a part.…”