Using herbarium samples for NGS methods – a methodological comparison

Herbaria harbor a tremendous amount of plant specimens that are rarely used for plant systematic studies. The main reason is the difficulty to extract a decent quantity of good quality DNA from the preserved plant material. While the extraction of ancient DNA in animals is well established, studies including old plant material are still underrepresented. In our study we compared the standard Qiagen DNeasy Plant Mini Kit and a specific PTB-DTT protocol on two different plant genera (Xanthium L. and Salix L.). The included herbarium material covered about two centuries of plant collections. A selected subset of samples was used for a standard library preparation as well as a target enrichment approach. The results revealed that PTB-PTT resulted in higher quantity and quality regarding DNA yield. For relatively recent herbarium specimens, and despite the lower overall yield of DNA, the Qiagen Kit resulted in better sequencing results regarding the number of filtered and mapped reads. We were able to successfully sequence a sample from 1820 and conclude that it is possible to include old herbarium specimens in NGS approaches. This opens a treasure box for phylogenomic research.


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Molecular biodiversity research as well as phylogenomic studies rely on a good, 36 comprehensive sampling. However, very frequently the required material is either not 37 available, e.g., in case of extinct species, or not accessible, e.g., if species occur in very 38 remote areas. To overcome the problems of insufficient sampling, herbarium specimens could 39 be used as a source of information (1,2). Herbaria harbor a massive amount of specimens that 40 were collected over several centuries and can thus be treated as treasure troves for 41 biodiversity research (3-7). It is estimated that around 70,000 new species are already housed 42 in herbaria, "waiting to be described" (6). However, although herbarium vouchers are a 43 valuable source of information, using them for molecular studies remained challenging (2,8). 44 The DNA of herbarium samples is usually highly degraded and fragmented and 45 extracting DNA from old tissues remains difficult. Mainly because of both, generally limited 46 success of DNA extraction and the challenges associated with PCR-amplification of highly 47 degraded DNA, researchers avoid to include historical specimens (2). In Sanger sequencing 48 times, amplification and sequencing required long, intact DNA fragments, and therewith 49 incorporating historical samples, especially from plants, was almost impossible. In contrast, 50 more recent developments in sequencing techniques enabled researchers to include 51 fragmented DNA (=short fragments) in their approaches (7,9). Nevertheless, a certain level of 52 DNA quality and quantity is necessary to include historical material in studies using NGS 53 methods. 54 For most phylogenomic studies, the DNA is usually extracted from fresh or silica 55 dried plant material by using a commercial DNA extraction kit. Historical samples require 56 more advanced methods with special regard to shorter fragment length and putative for freshly collected plant material followed by immediate drying in silica gel or freezing 66 (1, 13,14). 67 The first studies on ancient or archival DNA (aDNA) from plants were published in 68 the early 90s of the last century and dealt with plant remains in archaeological sites (e.g., 69 (15,16) among others). Studies dealing with DNA extraction from old herbarium samples 70 have used one of the following approaches: i) Early studies on herbarium material aiming at 71 sequencing single markers, e.g., ITS, simply used standard CTAB protocols for extraction 72 (17) or a modified version of it (12,18-21); ii) commercial kits were used with few 73 adaptations, e.g., increasing incubation times (18,19,22,23); or, iii) more specific protocols for 74 aDNA extraction were applied (e.g., optimized to obtain short sequences and to increase the 75 proportion of endogenous DNA in the extracts; (10,12,13,24). Since then, more and more 76 studies included historical plant material in phylogenomic studies (7,23,25,26). 77 However, specific protocols for aDNA are generally more expensive, time consuming and 78 require specific facilities and hygiene rules, not always available in systematic botany 79 laboratories. Moreover, extraction protocols were often optimized for a certain taxonomic 80 group or model organism (27).

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Although a few recent studies focused on comparing the efficiency of CTAB-based 82 extraction protocols with commercial kits (19), or comparing CTAB extractions with 5 83 protocols specific for aDNA (10), no studies yet have investigated the circumstances in which 84 aDNA methods (e.g., the PTB-DTT protocol described in (28) For each sample about 10 mg of leaf material was removed from the herbarium sheet 118 and transferred to an Eppendorf tube. The material was pulverized using a TissueLyser II 119 (Qiagen, Venlo, Netherlands). PTB-DTT extractions were done as described in Dabney et al.    2.552x10 -8 ; Fig 1A). Results are slightly correlated with the age of the herbarium specimen 240 (Pearson's r = 0.34 and r = 0.30 for the PTB-DTT and the Qiagen kit, respectively; Fig 1B).

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When treating the two genera separately, results were similar to those presented above.   A high-quality DNA shows a A260:A280 ratio of 1.8 and a A260:A230 ratio above 2. 263 Our results revealed that the DNA quality was overall higher for the PTB-DTT extractions.

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The results of the A260:A230 ratios could not be statistically compared, because the groups 267 show a significant heterogeneity of variances (Levene test, p-value = 0.00012) (Fig 2B).  Table 1  Effect of specimens' age on yield and quality 303 In the presented study, we extracted archival DNA of 37 herbarium specimens, with and quality than in Salix, especially for the old herbarium specimens (see Table 1 for details).  continue with the latter (more cost-effective) one.

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In Xanthium, 55%-65% of the reads mapped to the target regions of the baits kit. recovered. However, the library preparation differed from the simple skimming approach. The 428 assembly of the plastomes was done based on off-target reads of a target enrichment dataset.

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In these circumstances, to assemble complete plastomes might be difficult, and focus on the Mini Kit extraction. Our study showed that it is possible to include herbarium samples from 443 the last two centuries in NGS approaches by using standard commercial DNA extraction, 444 library preparation and target enrichment kits. However, in cases of challenging material (e.g., 445 old samples containing many secondary compounds, as is the case in genus Salix) or valuable 446 and rare material (e.g., type material, and/or scarce herbarium sheets) it might be preferable to 447 use specific aDNA extraction protocols.