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Comparative Analysis of SNP Discovery and Genotyping in Fagus sylvatica L. and Quercus robur L. Using RADseq, GBS, and ddRAD Methods

Department of Genetics, Faculty of Biological Sciences, Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz, Poland
Authors to whom correspondence should be addressed.
Academic Editor: Tadeusz Malewski
Forests 2021, 12(2), 222;
Received: 26 January 2021 / Revised: 9 February 2021 / Accepted: 11 February 2021 / Published: 15 February 2021
(This article belongs to the Special Issue Forest Genomics and Transcriptomics)


Next-generation sequencing of reduced representation genomic libraries (RRL) is capable of providing large numbers of genetic markers for population genetic studies at relatively low costs. However, one major concern of these types of markers is the precision of genotyping, which is related to the common problem of missing data, which appears to be particularly important in association and genomic selection studies. We evaluated three RRL approaches (GBS, RADseq, ddRAD) and different SNP identification methods (de novo or based on a reference genome) to find the best solutions for future population genomics studies in two economically and ecologically important broadleaved tree species, namely F. sylvatica and Q. robur. We found that the use of ddRAD method coupled with SNP calling based on reference genomes provided the largest numbers of markers (28 k and 36 k for beech and oak, respectively), given standard filtering criteria. Using technical replicates of samples, we demonstrated that more than 80% of SNP loci should be considered as reliable markers in GBS and ddRAD, but not in RADseq data. According to the reference genomes’ annotations, more than 30% of the identified ddRAD loci appeared to be related to genes. Our findings provide a solid support for using ddRAD-based SNPs for future population genomics studies in beech and oak.
Keywords: restriction-site associated DNA sequencing; SNP genotyping; technical replicates; population genomics; European beech; pedunculate oak restriction-site associated DNA sequencing; SNP genotyping; technical replicates; population genomics; European beech; pedunculate oak

1. Introduction

In the past decade, the development of the next-generation sequencing (NGS) methods combined with various types of newly developed genomic library preparation protocols provided the tools for relatively inexpensive discovery and genotyping of large numbers of loci useful in population genomics studies [1,2,3,4]. The ultimate way of obtaining genomic data from multiple samples is to apply whole-genome sequencing (WGS). This approach maximizes the quantity of information gathered, and opens up the possibility of a wide variety of analyses; however, it is currently prohibitively expensive and computationally challenging [5], especially in non-model species with large genomes. Analyses of genomic variations relevant in most population genomic studies can be conducted with reduced representation genomic libraries (RRL) [6,7] where only a few percent of the genome is sequenced. The most popular techniques use restriction enzymes to prepare the DNA (restriction-site-associated DNA: RAD) for sequencing. In recent years many methods based on the RAD approach were developed, differing in the number of enzymes used or in additional steps of library preparation [8].
In this study, we have focused on three RRL methods: RADseq [9], GBS (genotyping-by-sequencing) [10], and ddRAD (double-digest RAD sequencing) [11,12,13]. In RADseq, DNA is digested with a single, frequently cutting restriction enzyme. To such prepared DNA, barcodes and common adaptors with the first of two primer (P1) are ligated, then samples are pooled, randomly sheared, size-selected within a 300–700 bp window and, finally, in the final step P2 adaptors are ligated. Only the DNA containing both adaptors is PCR-amplified and sequenced in the single-end mode [9]. GBS is a simplified protocol also relying on a single restriction enzyme. Barcoded adaptors and common adaptors are randomly ligated to digested DNA, and fragments from multiple samples are pooled. Short DNA reads with both adaptor types are amplified and sequenced in single-end mode [10]. The ddRAD protocol uses two restriction enzymes—a rare and a frequent cutter. The first is used to generate fragments, after which barcoded primers are annealed. The second enzyme is used as the replacement for random shearing to improve the size selection step. Finally, P2 primers are ligated, and the fragments are amplified and sequenced afterward in double-end mode [11].
Precision and repeatability of genomic data are essential in most types of population genomics analyses, particularly in genomic selection surveys [14]. However, the ability to accurately genotype SNP loci from restriction-enzyme-based sequencing is a major concern [8]. Problems occurring in the genotyping step can be categorized into two groups: missing data and genotyping errors. These problems may originate from incorrect library preparation and un-optimized bioinformatics processing [13]. When ignored, these biases might affect the inferences of downstream analyses, to an unpredictable degree [15,16]. Optimizing library preparation and sequencing [13] mitigates sequencing errors. Unfortunately, some types of problems are unavoidable with restriction-enzyme-based methods, especially when the DNA quality is low [8,15].
On the other hand, sequencing artifacts can be minimized to some degree at the data preparation and core bioinformatics stage [17,18,19]. Current bioinformatics tools for genotyping from RAD data follow a series of three crucial steps: (1) raw data processing (preparation); (2) reading of the alignment against a reference genome or de novo assembly of the sequence tags; and (3) variant calling and filtering [13,20]. These steps aim to provide reliable and precise data; however, due to the probabilistic nature of the bioinformatic algorithms, they can alleviate errors while also generating some new mistakes that can have a profound effect on the final results of downstream analyses [21]. However, overly conservative filtering of genomic SNP data may cause data loss leading to misestimation of genetic effects [12].
One of the main advantages of using RAD methods for genotyping of non-model species is that loci can be identified de novo, without the availability of a reference genome [8]. However, the presence of reference genomes may be beneficial as RAD loci predicted in combination with a reference genome will be useful in filtering SNPs from paralogous or repetitive sequences, identifying indel variation, and avoidance of calling wrong loci due to biological contaminations [8,22]. With well assembled and annotated reference genomes, identified RAD loci can be readily positioned along the genome and may become directly useful in association studies. However, only a few studies have compared the efficiency and precision of SNP discovery and genotyping based on de novo versus reference-aligned approaches to date [23,24,25].
Regardless of the nature of any genotyping problems, one way to monitor the levels of inconsistencies in SNP discovery and genotyping is to use technical replicates [13,19] or include parent-offspring dyads, if permitted by experimental designs [23]. Mastretta-Yanes et al. [19] defined several types of possible errors that could be investigated based on the use of technical replicates while varying different parameters in Stacks software [26]. Using ddRAD libraries, they analyzed a non-model plant species without a reference genome. The study demonstrated that with technical replicates, it is possible to optimize and tune a de novo genotyping pipeline and to identify and mitigate sources of errors [19].
Due to their foundation roles in terrestrial ecosystems and their broad economic importance, forest tree species have been thoroughly investigated in the areas of population genetics and evolutionary biology. The accumulated genomic resources, including genome assemblies of major tree taxa (Populus trichocarpa [27], Eucalyptus grandis [28], Pinus taeda [29], Olea europea [30], and Quercus lobata [31]), accelerated the progress of population genomics in forest trees. The number of studies involving genomic data is continuously increasing, including works based on RAD approaches [22]. Forest trees, due to their characteristic life traits [32] are highly heterozygous as compared to other species [33]. The high level of genome-wide heterozygosity may complicate genome assemblies [31] and confound SNP discovery and genotyping in RAD-based experiments. However, studies aimed at optimizing RAD approaches in forest trees are limited [22].
In this study, we investigated the efficiency of SNP discovery and genotyping in two of the most important broadleaved tree species in Europe, namely common beech (Fagus sylvatica L.; abbr. FS) and pedunculate oak (Quercus robur L.; abbr. QR), both belonging to the Fagaceae family. Using the same set of individuals within the species, we evaluated the three RAD approaches mentioned earlier: RADseq, GBS, and ddRAD. Taking advantage of the reference genomes of beech and oak, we contrasted de novo and reference-aligned marker discovery approaches. Among the samples of each species, we included technical replicates (four individuals sampled twice), which enabled us to monitor the replicates genotyping consistency while optimizing specific parameters of the applied bioinformatics pipelines. Our ultimate goal was to fine-tune protocols and the approaches for gathering as many loci as possible with the fewest possible artifacts and the lowest proportion of missing loci. We believe that our findings will be useful for selecting the most appropriate RAD approaches for population studies in beech and oak, and will provide best-practice guidelines for processing RAD data in general.

2. Materials and Methods

2.1. Species Background

Common beech (Fagus sylvatica L.) and pedunculate oak (Quercus robur L.) are broadleaved, monoecious, wind-pollinated, and highly outcrossed tree species [34,35], both belonging to the Fagaceae family. In Europe, they play essential roles in forest ecosystems’ ecology and are essential in the forestry-based economy in several countries [36,37,38]. Genetics is one of the most thoroughly researched aspects of beech and oak [39,40]. However, while it is continuously growing, the genomic-based knowledge of these two species is still limited but continuously growing [41,42,43,44,45]. Reference genomes of the two species were published relatively recently. The common beech haploid genome (version: 1.3) [46] is the smaller of the two, totaling 542.3 Mbp with a GC-pair content of 35.69% and 62,085 predicted genes. The pedunculate oak haploid genome (version: PM1N) [47] is distinctly larger: 814.36 Mbp, consisting of 35.65% GC-pairs, and 25,808 well-defined genes.

2.2. Sample Collection

Beech individuals selected for this study were sampled from the provenance trial located in the Siemianice Experimental Forest District (51°12′52.4″ N, 17°59′50.1″ E) in south-central Poland [48]. The trial consists of 71 provenances originating mostly from Central Europe; however, for this study, we sampled 91 individuals representing 47 provenances. Among them, four individuals from four different provenances were sampled twice from the same individual and were considered technical replicates (duplicated samples) and marked thereafter as FA, FB, FC, and FD. Oak individuals originated from the provenance/family of a common-garden trial located in the Mogilica Forest District (53°12′36.7″ N, 15°13′39.7″ E) in north-western Poland [49]. The trial consists of the progeny of mother trees originating from eight Polish provenances (50 mother trees/provenance). As with beech, we sampled 91 individuals representing all of the provenances and 64 unique mother-trees families, and four individuals were selected as technical replicates, each one being the progeny of a different provenance/mother-tree, marked as QA, QB, QC, and QD. Such a wide sampling of individuals from several populations was intended to account for the possibility of large genetic diversity within species.
Replicates were processed separately to assess the repeatability of SNP identification and calling procedures. Altogether, within each species and method, 95 samples were subjected to DNA isolation and sequencing (570 samples in total). Samples for GBS and RADseq libraries were collected in 2014 from the same individuals. However, to generate ddRAD libraries, the trees were sampled in 2015, and some individuals (not the replicates) were replaced due to health-related dropout cases. Details regarding the sampled material is presented in Tables S1.1 (for beech) and S1.2 (for oak).

2.3. DNA Isolation, Library Construction, and Sequencing

The sequencing of reduced-representation libraries demands high molecular weight DNA >50 kb, with a concentration >30 ng/µL and total mass of 1.5–3 µg [50]. To meet these requirements, we collected flushing leaf buds. Sampled material was dried to ≈10% humidity with a phytotron (BINDER WTC KB 240) for 24 h at 30 °C; afterwards, 30 mg of tissue was stacked in sterile 2 mL Eppendorf tubes and ground at 30 Hz in a laboratory mill (Mixer Mill MM 400, Retsch, Haan, Germany). DNA was isolated using GeneMATRIX Plant & Fungi DNA Purification Kit (EURx, Gdańsk, Poland), with slight modifications to the manufacturer’s protocol (for details, see Supplementary Materials).
Isolated DNA was shipped to external service providers for enzyme optimization, library construction, and sequencing: GBS—Cornell University (Ithaca, NY, USA); RADseq—Floragenex Inc. (Portland, OR, USA); and ddRAD—IGA Technology Services (Udine, Italy). Details of library construction and sequencing are presented in Table 1.

2.4. Data Processing and Analysis

Sequence datasets were checked with FastQC software (v0.11.8; [51]) to assess the average Phred quality score of each nucleotide position, and the presence of artifacts such as sequencing adaptors. Samples were demultiplexed using the process_radtags tool: a part of the Stacks pipeline (v2.3) [26]. Process_radtags was configured to discard reads with average Phred-score <20, remove the barcode and trim 3′-ends of the reads if necessary. Among reads from the GBS library, the presence of contaminants (e.g., Illumina universal adaptors) and a quality drop at the 3′-end meant that these samples were trimmed to 64 bp. RADseq and ddRAD data were free from these artifacts, providing 90 bp and 110 bp reads, respectively. Quantity and quality of reads of each individual were again checked with FastQC; the results were summarized with MultiQC (v1.7) [52]. Finally, the reads were processed for additional purification with Trimmomatic [53] using default settings.
Initial analyses were conducted based on technical replicates. The intention behind the use of technical replicates was to investigate to what degree the genotypes reported from two replicates of the same individual are identical and how the parameters used in de novo or reference-based SNP calling affect that similarity. For the de novo approach, we used Stacks (v2.3) [26] to construct loci and extract SNPs. In this study, we tested a set of three parameters, similarly to the study of Mastera-Yanes et al. [19]. The core parameters in the denovo_map pipeline were varied across the course of the experiment: m—a minimum number of reads necessary to create a stack (range 2 ÷ 15, default 3); M—maximum number of mismatches between stacks while searching for the allele in an individual (range 2 ÷ 10, default 2); and n—the maximum number of mismatches between loci searched in a joint pool (range 1 ÷ 5, default 1). Only one parameter at a time was changed, while others were set to the default values. The results were outputted to ‘vcf’ file format using the built-in ‘populations’ pipeline, providing 28 datasets for each species and the library for further analyses (168 datasets in total).
In the reference-based approach, we used bwa software (v 0.7.17) [54] with default settings to map the reads against available reference genomes and tested the effects of varying different parameters on the outcome of the SNP-calling procedure. This step was carried out using Heap software (v0.8) [55]. The program uses an approach similar to GATK [56], but it is less stringent and has a rather simpler setting. The following parameters were tested: depth—a minimum depth of filtered reads that support each of the reported alleles at the individual level (range 3 ÷ 10, default 3), and mapq—the posterior probability that mapping position is wrong, expressed as a Phred score (20, 30, 40; default 20). Similarly to the method outlined above for de novo analyses, only one parameter was changed at a time while other parameters were fixed to the default values. The results were outputted to a vcf file format, delivering 34 datasets for each species and the library for further analyses (234 datasets in total).
The results obtained for technical replicates were filtered with vcftools (v0.1.16) [57] to exclude indels and include only biallelic SNPs present in at least 6 out of 8 samples (75%). The key indicators and error types (Table 2) were calculated for each replicated pair in each dataset, and averaged with a custom-designed script written in bash. Note that due to filtering out of anything other than biallelic loci, the allele errors of type A|C-A|T (as indicated in [19]) could not be assessed in our study.
Finally, we analyzed the full dataset to assess the efficiency of different RRL libraries used in this study. To reduce redundancy in the full datasets, we have included only one replicate from a pair: the one with the higher read number. Samples with less than 50% of the average number of reads were discarded from the analysis [19]. SNPs from de novo (Stacks) and reference-based (Heap) protocols were generated using default settings. The outputted results were filtered with vcftools [57] to exclude indels, non-biallelic SNPs, and markers present in less than 80% of the samples. To reduce linkage disequilibrium (LD) bcftools was used with r2 > 0.5 in a 1000 bp window. Filtered SNPs were analyzed to determine differences in the generated data from the GBS, RADseq, ddRAD genomic libraries and how genotyping strategies affect each of them.
Basic statistics of different datasets were generated with bcftools from the samtools package (v1.9) [58] with option: stats. A summary of the statistics was conducted in the R environment [59]. Annotation of the reference-based SNPs was carried out using SnpEff [60] using default settings, with gff3 files provided with the reference sequences of beech and oak [46,47]. The annotation was performed on the sets containing biallelic SNPs with markers present in at least 80% of the samples, and minor allele frequency (MAF) >0.05.

3. Results

3.1. Technical Replicates

The highest average number of reads after cleaning was obtained for GBS libraries for both species: FS: 1.93 × 106 reads/sample; QR: 3.34 × 106 reads/sample. In the other two libraries the quantity of data was considerably lower: RADseq—FS 1.15 × 106 reads/sample, QR: 1.15 × 106 reads/sample; ddRAD—FS: 1.07 × 106 reads/sample; QR: 1.08 × 106 reads/sample (for details, see Table S2). We noticed that the replicated pairs with higher read numbers generated more SNPs; however, larger differences in the number of reads between individuals within the pair negatively affected the quantity of data obtained per pair.
The analyses conducted using default settings showed that different types of libraries provided comparable results for oak and beech (Table 3). The ddRAD and RADseq datasets, in contrast to GBS, generated a proportionally higher number of raw SNPs in the mapping approach than in the de novo approach. We also found that after filtering (only biallelic SNPs; present in at least 6 out of 8 samples; indels removed), the set of SNPs generated de novo had a distinctly higher percentage of the retained markers than the set obtained using the mapping approach (average 53.4% vs. 43.2%; Table 3). However, the actual number of SNP loci after filtering was the highest for ddRAD, and from the mapping approach in particular (Table 3).
The proportion of good loci (GL) was significantly higher in GBS and ddRAD datasets as compared to RADseq (Table 3). This may have been caused by fragmention of DNA, as the RADseq protocol heavily relies on initial DNA quality and may produce uneven coverage among the samples [15,61]. This assumption is supported by the distinctly altered proportions of missing loci (ML) and allele error (LE) counts in the RADseq data as compared to the other two libraries. The proportion of missing alleles (MA) in the GBS and ddRAD sets increased when using the mapping method. This is probably a result of the SNP calling procedure, where the allele must be present in at least three reads to call a variant. In general, all libraries had similar levels of missing data (MD).
In conclusion, given the yield of filtered SNP loci and the proportion of reliable loci (GL) for both species, the most efficient method to gather the largest quantity of data is by applying the ddRAD technique and implementation of a mapping approach for SNP discovery and calling. Finally, in respect of ‘good’ loci, we were able to find 52,280 in beech and 67,977 in oak, given the filtering criteria applied.

3.2. Influence of Parameters Used in Genotyping Procedures

Finding the best-fitting parameters is a crucial aspect of bioinformatics [62]. By increasing the threshold for the minimum number of reads required to create a locus (m) in the de novo procedure using Stacks (v2.3) [26], the number of SNPs in all analyzed datasets was decreased. The parameter itself may be treated as an additional filtering tool for obtaining loci with higher coverage, and for increasing reliability of the results. Comparing the number of markers generated with a variable m value (for details see: Tables S3.1 and S3.2) indicated a decrease of SNPs reported in raw and filtered datasets, as expected. The share of good loci (GL) in all analyzed cases decreased with increasing m, which was correlated with the increase of missing loci (ML). In general, the m parameter had a minor effect on MA, LE, and MD; however, the filtering nature of the m value mitigated locus error (LE) to some degree in RADseq sets (for detailed results, see: Tables S3.1 and S3.2). Overall, we found the default value (3) of the m parameter to be the best in optimizing the quantity and quality of discovered SNPs for both species.
We found that the two Stacks parameters that allow for mismatches, namely M (number of mismatches between stacks within the individual) and n (number of mismatches in the joint pool of individuals), elevated the numbers of raw markers in all studied cases. Only in the QR-GBS dataset did the number of filtered SNPs slightly decreased with increasing M (Table S3.2). This occurs when a locus is underrepresented in a joint catalog being filtered for a minimum number of individuals, which causes a marker dropout. All datasets were insensitive in terms of the influence of the M parameter on the key indicators defined in Table 2. For example, we observed only a slight decrease (2.3%–4.9%) in the GL when increasing the number of mismatches (for detailed results, see: Tables S3.1 and S3.2). In general, our results indicated that the elevated number of mismatches within the individual has only a slight influence on the key indicators, and the most important factor responsible for marker quality is the initial quality of the data itself. However, when choosing the optimal value of M, several other factors must be considered, such as the length of sequence reads, the biology of species (including heterozygosity levels), and the degree of relatedness/differentiation among individuals or populations. Considering the above, and the fact that our sampling material in each species represented several populations (see Methods) we decided to choose the optimal value of the M value to be 2 (default), both for beech and oak, although slightly higher values should provide more markers with only a slight loss of GL.
The n parameter is responsible for allowing mismatches in a joint pool, and as expected, increasing n resulted in increased numbers of SNPs in both raw and filtered data. The GL parameter decreased slightly in GBS and ddRAD sets (<5%) and was rather stable in RADseq. The effect of n on other key parameters was considerably lower. The RADseq data was weakly affected by changes of n parameter (for detailed results, see Tables S3.1 and S3.2). When choosing the appropriate value for n parameter, similar cautions should be needed as for parameter M. However, it is also important to note the degree of diversity among populations/individuals included in the analysis. Since increasing n may also increase the proportion of paralogous loci we decided to use the default value (1) as the optimal value for this parameter in our study.
The Heap tool used in this study implements a similar bioinformatics approach as GATK to call SNPs, but it seems to be less conservative [55]. In Heap, the depth parameter sets a threshold for the minimum number of mapped reads/nucleotides needed to call an SNP. It can be treated as an additional filtering tool for increasing confidence in returning markers that are not sequencing errors. The impact of depth on key indicators is a consequence of loss of markers when an individual from a replicate pair has a lower number of reads. In general, increasing depth distinctly decreased the number of filtered SNPs; however, GL decreased slightly or remained unchanged. In most cases, the change of GL, along with depth, was negatively correlated with ML (for details, see Tables S3.3 and S3.4).
The mapq parameter is also quality-related parameter, although it did not influence the key indicators; rather, it had an impact on the number of markers generated (for details, see Tables S3.3 and S3.4). Across the datasets, those from GBS were affected most when increasing mapq. Using high mapq values is justified, especially when the Phred quality of the delivered data is low, likely generating false-positive SNPs. Considering the ‘filtering’ nature of both parameters when high-quality genome and reduced representation data is available, we recommend setting them to default values (depth = 3; mapq = 20).

3.3. Whole Datasets

In our analysis, we initially decided upon the threshold that an individual would need to have at least 50% of the average number of SNP per individual to be included in subsequent analyses. In beech, all 91 samples fulfilled this criterion; however, in oak, three samples in GBS and one in RADseq did not pass the threshold, and these were discarded from further analyses.
In general, there was a congruence between the replicated samples and the whole datasets in the numbers of SNPs retained after filtering (Table 3 and Table 4). The numbers of SNPs obtained based on the whole datasets depended on the genome’s size, except in the case of RADseq data. The filtering process increased the sample depth from 25.12 (unfiltered) to 34.35 (filtered), which seems to be the level acceptable for most population analyses. The Ts/Tv ratio was comparable across all datasets. However, after filtering, the average Ts/Tv ratio increased by 0.18 for the de novo method and only 0.05 for the mapping method. The increased Ts/Tv ratio in the filtered sets is the expected effect of higher SNP quality after filtering [63].
RADseq appeared to be the least efficient method for the identification and calling of SNPs. Despite the large initial number of loci, data filtering resulted in a dramatic reduction of loci that fulfilled the criteria. It seems that the most critical problem in RADseq is repeatability of loci identification. Many initial loci were filtered out due to their uniqueness (MAF >0.05) or a high level of missing data. This correlates the high proportions of MD and MA observed in the replicated samples. We suspect that the poor RADseq performance could have resulted from low quality of input DNA samples, which may have been attributable to errors made during the construction of libraries where some samples may have not been sufficiently cleaved with a restriction enzyme or fragmented by sonicators, thus causing the dropout of many RAD sites. SNPs’ final outputs based on designed filtering criteria exclude this type of library for population genomic analyses.
GBS provided reasonably large datasets for both beech and oak samples. A range of ~8000 to ~28,000 SNPs per genome (depending on species and genotyping method; Table 4) seem to be sufficient for most types of published population genomic studies; however, such density may not be adequate for in-depth association analyses. Assuming standard filtering (SNPs observed in >80% of individuals; MAF > 0.05; no LD), GBS provided more data using the de novo approach than the mapping method. This is likely related to the size of the sequence reads of GBS data. Since some shorter reads could be mapped to multiple genome positions, they were discarded as ambiguous and not considered for identification of SNPs. However, the difference in numbers of SNPs between de novo and mapping methods was minimized when SNP loci were required to be observed in all sampled individuals (SNPs: 100%), which resulted in reduced numbers of SNPs. This suggests that, for GBS, the mapping method is more conservative than the de novo approach when one allows for some level of missing data to be acceptable for downstream analyses.
The ddRAD approach appeared to be the best choice for identification and calling of SNPs in both beech and oak. After filtering, this method returned ~28,000 and ~35,000 SNPs for beech and oak, respectively. Interestingly, the locus counts were similar for de novo and mapping methods of SNP identification, which should be considered a reciprocal confirmation of the data quality. Even when requiring that a SNP locus must be genotyped among all sampled individuals (i.e., no missing data), and setting no MAF limits, there were still ~34,000 and ~53,000 markers available for beech and oak, respectively (Table 4). Such numbers of fairly repeatable SNP markers satisfy most types of population genomic studies, and should be helpful even in some genomic association analyses.
Using the available reference genomes (beech—[46]; oak—[47]), we have annotated the filtered SNPs obtained from the mapping approach to check their genomic position and usefulness in future studies. In both species, a similar pattern was observed; however, some differences between these species may result from differences in the genome annotation methods. In both cases, the GBS markers were mostly (>75%) associated with non-gene sites, while in RADseq and ddRAD a considerable proportion of SNPs was related to genes (Table 5). However, about 26% of SNPs in beech and 22% in oak were located in introns or were synonymous sites (Table 5). Considering the total number of SNPs in the filtered data sets related to genes, but excluding intron and synonymous variants, we obtained the best results with ddRAD, indicating 4105 and 3658 SNP loci in beech and oak, respectively (Table 5).

4. Discussion

In recent years, a growing interest in reduced-representation genomic approaches and their applications proved their usefulness in numerous studies [8,64,65]. However, the variety of available library types and analytical pipelines to process this data can be confusing, especially for researchers with limited experience, despite the broad support of research community. The use of technical replicates in the optimization of SNP identification and calling is described in many studies [19,66,67,68] and it appears to be a good practice to tune the pipeline parameters to the species and scope of the study, and to monitor the quality of SNP identification. Our results confirm these findings and provide additional information in the discussion on how different types of libraries from the same species can be influenced by distinct genotyping strategies.
Testing of technical replicates is inexpensive, and it should be the initial requirement when using RAD-based genomic libraries for marker discovery. The optimization step can provide initial insights into the expected range of results in a larger study, and reduce risk of project failure. The analytical process after data delivery is fast, and evaluation of a few samples on a standard computer (e.g., four-thread CPU with 8 GB of RAM) takes from 24 to 72 h. Due to the rapid development of out-of-the-box solutions, both offline (e.g., dDocent [61]) and online (e.g., Galaxy [69]), the data analysis process can be performed by staff even only moderately involved in the field of bioinformatics. However, to obtain informative SNPs, specific requirements for the isolated DNA must be met [70].
NGS is particularly sensitive to low DNA quality, and vulnerable to errors which can emerge during the library preparation [71,72]. Our results suggest that laboratory errors can lead to genomic region/marker dropout, as in the case of our RADseq dataset, despite a satisfying number of reads per sample. However, RADseq is known to suffer from large proportions of missing data [73]. When outsourcing the construction of libraries and subsequent sequencing, research teams should be focused on providing good quality DNA or fresh raw material to avoid potential data errors [74].
Overrepresentation in de novo SNP data, as observed in GBS datasets, can result from differences in genome size among individuals within the species [75,76], especially when using a restriction enzyme that cuts frequently, as illustrated in our GBS data. When the mapping approach is used, sequencing errors may decrease SNP numbers after unfitting bases are discarded. Nonetheless, this reference-based strategy would provide data that may be more comparable with that of other studies, and it can also deliver additional information e.g., annotation of SNPs [8]. This genotyping strategy should always be the first choice when a reference genome is available [77]. On the other hand, although the availability of plant genomes is increasing on a daily basis, they are still scarce. In the absence of a reference genome, de novo genotyping is a reliable alternative, as demonstrated in other studies [78].
It should be noted that in this study, we used an initial filtering of SNP data (SNPs biallelic; present in 75–80% of samples; indels removed), which generally alleviates some common genotyping problems, including missing loci. In particular, the choice to filter out those loci present in less than a specified fraction of samples (e.g., 80%) appears to be an efficient way of pre-selecting reliable loci [8,79]. Therefore, our variation of Stacks or Heap parameters had only a moderate effect on the size and quality of resulting SNP datasets. After the initial optimization, based on replicated samples, the choice of the correct parameters to genotype the whole sample set is always the trickiest part of the study. Here, we would not like to provide unequivocal information on what exact values should be chosen, because these depend on the scope of analysis, the species’ biology, and even sampling strategy (local or wide sampling). However, we intend to share some observations and guidelines on how to perform the optimization process.
  • The assessment of the raw and filtered numbers of markers (i.e., biallelic SNPs, present in at least 6 out of 8 samples, indels removed) helps to detect library/data errors. For example, significant loss of markers in a filtered set will usually be a signal of uneven genome coverage by reads, regardless of the genotyping method.
  • When assessing the number of markers reported based on parameters associated with more restrictive genotyping (e.g., m, depth, mapq), first check the number of SNPs after filtering (with the abovementioned criteria). In some sets, more stringent filtering did not cause the expected improvement but rather the possible loss of SNPs.
  • Pairs of replicated samples from the same individuals will always share a significantly higher proportion of good loci (GL) with each other than with any other sample. This observation can be used as a quality control tool to determine whether a swap of samples occurred.
  • To overcome a threat of false positive SNPs, which can occur with highly elevated numbers of markers, we suggest focusing on the proportion of good loci as the most important key indicator.
  • Increasing the minimum number of reads necessary to create a stack (m) will always decrease read number and cause dropout of underrepresented makers leading to the decreased levels of GL and a higher proportion of ML (as in the case of GBS and ddRAD) and, in cases of problems with data uniformity throughout the whole genome, shifted MA values (as in the case of RADseq).
  • If a reference genome for the species under analysis is available, optimization should be conducted using the reference-based approach. It can be expected to deliver results that are more reliable, and more comparable to those of other studies.
  • The influence of SNP-calling procedure has a profound effect on the number and quality of markers. Both depth and mapq should be treated as filtering tools; high values for these parameters will significantly decrease the number of markers returned. If more stringent filtering is necessary, using elevated mapq is a preferable option due to having no effect on key indicators. These symptoms are usually a sign of increased detection of false positive SNPs [19].
  • Shifted mismatch both on an individual level and in the joint catalog should be adjusted with respect to species biology and the sampling strategy applied [66].

5. Conclusions

Genotyping by next-generation sequencing (NGS) of reduced-representation genomic libraries associated with restriction enzymes became a common approach to identify large numbers of genetic markers (mostly SNPs) uniformly distributed across genomes. However, the number and quality of RAD-based markers obtained in particular studies depends on many aspects, including the quality of DNA isolation, the choice of RRL, the type of restriction enzymes, the design of sequencing (resulting in sequencing depth), and the bioinformatics pipelines used for identification and calling of SNPs [8,22]. Testing all of the possible variables is beyond the scope of a single study. In this paper, we briefly evaluated the three RRL approaches (GBS, RADseq, ddRAD) and different methods of SNP identification (de novo or by reference genome mapping) to find the best toolset for future population genomics studies in two broadleaved tree species: F. sylvatica and Q. robur.
We found that the most promising approach—providing relatively large numbers of reliable SNPs—is to employ the ddRAD technique and a calling approach based on mapping sequence reads to a reference genome. Based on about 90 individuals within species, we found ~28,000 and ~36,000 loci for beech and oak, respectively, given typical filtering criteria (MAF > 0.05; SNPs present in >80% samples; LD r2 < 0.5). However, when relaxing LD filtering limitations, these numbers increased up to ~56,000 and ~59,000 respectively (Table 4). Based on technical replicates, we estimated that in ddRAD more than 80% of SNP loci should be considered reliable. Additionally, according to annotations on the reference genomes, we found that in both species more than 30% of the identified loci could be related to genes. These findings provide a solid support for the use of ddRAD-based SNPs for future population genomics, or even for genomics selection studies.

Supplementary Materials

The following are available online at, DNA isolation protocol, Table S1.1.—European beech (Fagus sylvatica L.) sample basic information; Table S1.2.—Pedunculate oak (Quercus robur L.) sample basic information; Table S2. Basic replicate samples information; Table S3.1. Results of parameters (m, M, n) altering for genotyping with stacks (de novo method) of European beech (Fagus sylvatica L.) replicated samples; Table S3.2. Results of parameters (m, M, n) altering for genotyping with stacks (de novo method) of pedunculate oak (Quercus robur L.) replicated samples; Table S3.3. Results of parameters (depth, mapq) altering for genotyping with Heap (mapping method) of European beech (Fagus sylvatica L.) replicated samples; Table S3.4. Results of parameters (depth, mapq) altering for genotyping with Heap (mapping method) of pedunculate oak (Quercus robur L.) replicated samples.

Author Contributions

Conceptualization, B.U. and J.B.; Methodology, B.U. and J.M.; Investigation, B.U. and J.M.; Data curation, B.U.; Writing—original draft preparation, B.U.; Writing—review and editing, B.U., J.M., and J.B.; Supervision, J.B.; Project administration, J.B.; Funding acquisition, J.B. All authors have read and agreed to the published version of the manuscript.


The study was supported by the National Science Center, Poland (2012/04/A/NZ9/00500), and the Polish Ministry of Science and Higher Education under the program “Regional Initiative of Excellence” in 2019–2022 (grant no. 008/RID/2018/19).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw GBS, RADseq and ddRAD data have been submitted to NCBI SRA database under the BioProject ID number PRJNA694960.


We would like to thank Władysław Barzdajn from Poznań University of Life Sciences and the staff of Experimental Forest District in Siemianice as well as Roman Rożkowski from the Institute of Dendrology, Polish Academy of Science and the staff of the Choszczno Forest District for their support on the study sites. We also to thank our lab team members: Ewa Sztupecka and Katarzyna Meyza for their outstanding job in fieldwork and DNA isolations.

Conflicts of Interest

The authors declare no conflict of interest.


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Table 1. Summary of enzymes and sequencing modes used for each library type.
Table 1. Summary of enzymes and sequencing modes used for each library type.
Library TypeEnzyme and SpeciesSequencerRead LengthSequencing ModeBarcode Length (bp)
GBSEcoT22I—bothHiSeq 2000100Single-end5–8
RADseqPstI—bothHiSeq 2000100Single-end10
ddRADSphI/Sau3AIF. sylvatica
PstI/Sau3AIQ. robur
HiSeq 2500125Paired-end15
Table 2. Key indicators used to assess method efficiency.
Table 2. Key indicators used to assess method efficiency.
IndicatorDescriptionExamples *
Good loci (GL)Genotypes in both replicates are the same.R1 A|A—R2 A|A
R1 A|C—R2 A|C
Missing allele (MA)A variant of one genotype partially fits the otherR1 A|A—R2 A|C
Locus error (LE)Both genotypes differ with no common allelesR1 A|A—R2 C|C
Missing loci (ML)One genotype available, second is absent.R1 A|A—R2 0|0
Missing data (MD)Both genotypes of a replicate are absent.R1 0|0—R2 0|0
* R1/R2—first and second technical replicates for a pair; A,C—example nucleotides; 0|0—missing genotype.
Table 3. Total number of raw (unfiltered) and filtered SNPs, and key indicators for replicated samples and each library type and genotyping method; data generated with default settings (GL—good loci, MA—missing allele, LE—locus error, ML—missing loci, MD—missing data).
Table 3. Total number of raw (unfiltered) and filtered SNPs, and key indicators for replicated samples and each library type and genotyping method; data generated with default settings (GL—good loci, MA—missing allele, LE—locus error, ML—missing loci, MD—missing data).
Fagus sylvaticaQuercus robur
Average number of reads/sample1,927,3171,159,3601,071,3693,342,0971,513,0531,086,261
Genotyping methodde novomapde novomapde novomapde novomapde novomapde novomap
Number of raw SNPs32,18021,86037,06343,53881,338145,54867,12351,74745,02688,15176,376162,674
Number of SNPs after filtering *20,81312,66616,45413,76442,73265,08242,85929,48210,88813,41551,81478,786
% of SNPs after filtering *64.6857.9444.3931.6152.5444.7263.8556.9724.18%15.22%67.8448.43
Key indicators of the filtered SNPsGL87.01%84.80%46.23%48.43%80.90%80.33%87.50%86.70%42.60%46.00%87.25%86.28%
* Only biallelic SNPs, present in at least 6 out of 8 samples, indels removed.
Table 4. Basic statistics of results generated with de novo (Stacks) and map (HEAP) approaches on the whole datasets. (MAF—minor allele frequency; LD—linkage disequilibrium; TV/TS—transition to transversions ratio).
Table 4. Basic statistics of results generated with de novo (Stacks) and map (HEAP) approaches on the whole datasets. (MAF—minor allele frequency; LD—linkage disequilibrium; TV/TS—transition to transversions ratio).
Fagus sylvaticaQuercus robur
de novomapde novomapde novomapde novomapde novomapde novomap
Number of samples919191919191878790909191
Unfiltered dataNumber of SNPs124,34958,788157,755130,325391,161482,198325,260180,214257,606304,664406,455634,434
TS/TV ratio1.571.511.081.171.551.811.881.901.331.521.381.75
Avg. sample depth34.130.728.524.416.715.922.622.428.726.525.925.0
Filtered data
SNPs > 80% + MAF >0.05 + removed LD
Number of SNPs16,81682703071108328,54128,19928,90715,91970923035,24536,058
% of SNPs retained13.52%14.07%1.95%0.83%7.30%5.85%8.89%8.83%0.28%0.08%8.67%5.68%
TS/TV ratio1.501.511.401.591.621.742.192.281.591.281.561.58
Avg. sample depth40.535.438.240.821.921.426.329.447.746.432.831.4
Alternative filters with no LD filteringSNPs >80%39,95324,21411,658289287,832124,769100,57870,5504033926132,158172,450
SNPs > 80% + MAF > 0.0520,04311,7603707145141,17756,28833,53022,09484740245,84159,475
SNPs 100%10,23797369415,09234,75114,81316,21919531,56653,639
SNPs 100% + MAF > 0.054769463901653916,2274052470411919217,284
Table 5. Categories of filtered (SNPs > 80%, MAF > 0.05, LD removed) and annotated SNPs.
Table 5. Categories of filtered (SNPs > 80%, MAF > 0.05, LD removed) and annotated SNPs.
Fagus sylvaticaQuercus roburFagus sylvatica (%)Quercus robur (%)
3 prime UTR variant165356875514542.00%3.23%2.44%0.35%0.42%1.26%
5 prime UTR premature start codon gain variant11549--430.13%0.46%0.17%--0.12%
5 prime UTR variant23142425-2600.28%1.29%0.86%0.03%-0.72%
Initiator codon variant--3-----0.01%---
Missense variant29820526901802725213.60%18.93%9.54%1.13%11.44%6.99%
Missense variant&splice region variant93417-400.11%0.28%0.15%0.04%-0.11%
Splice acceptor variant&intron variant31271-40.04%0.09%0.10%0.01%-0.01%
Splice donor variant&intron variant3-151-40.04%-0.05%0.01%-0.01%
Splice region variant3112--50.04%0.09%0.04%--0.01%
Splice region variant&intron variant371317721-2430.45%1.20%0.63%0.13%-0.67%
Splice region variant&synonymous variant61314-370.07%0.09%0.11%0.03%-0.10%
Start lost--41-4--0.01%0.01%-0.01%
Stop gained8711481360.10%0.65%0.40%0.05%0.42%0.10%
Stop gained&splice region variant--4--1--0.01%--0.00%
Stop lost1-8--10.01%-0.03%--0.00%
Stop lost&splice region variant-----2-----0.01%
Stop retained variant1-1--30.01%-0.00%--0.01%
Synonymous variant25320119631452526533.06%18.56%6.96%0.91%10.59%7.36%
Intron variant12041253418123427606614.56%11.54%12.12%7.75%11.44%16.82%
Intergenic region83073259710,0158312,21210.04%6.74%9.21%62.91%35.17%33.87%
Downstream gene variant20331786249204637634324.58%16.44%22.16%12.85%15.68%17.59%
Upstream gene variant33822219867219635512640.89%20.41%34.99%13.79%14.83%14.22%
Non genes624547218,71314,25715523,68175.51%43.58%66.36%89.56%65.68%65.67%
Genes 202561194861662811237724.49%56.42%33.64%10.44%34.32%34.33%
Genes—excluding intron and synonymous56828541052832936586.87%26.32%14.56%1.78%12.29%10.14%
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