2.1. Plant material and DNA extraction

Silica-gel dried leaf tissue from two accessions of L. vulgare and L. virgatum, collected during field excursions, was used for the present study (for voucher details see S1 Table). Genomic DNA (gDNA) was extracted following a CTAB extraction protocol adapted from [27] and [28] with several minor modifications, including the additional use of phenol during nucleic acid purification, and the use of sodium acetate instead of ammonium acetate as well as an additional isopropanol step during alcohol precipitation. Each extraction was quality-checked via agarose gel electrophoresis, and DNA concentration and purity was determined using a NanoDrop spectrophotometer One (Thermo Fisher Scientific, Waltham MA, USA).

2.2. Amplification via long-range PCR

Chloroplast DNA was amplified using primer pairs by Uribe-Convers et al. (2014) [29] to generate 16 overlapping PCR fragments spanning the whole chloroplast. Before amplification, we assessed their potential in Anthemideae by fitting them onto Artemisia frigida (available with the ID NC_020607.1 in Genbank). This was done by performing megaBLAST searches [30] at the National Center for Biotechnology Information (NCBI) BLAST website https://blast.ncbi.nlm.nih.gov/Blast.cgi [31]. Based on the estimated product sizes and overlap lengths in A. frigida, for fragments 2–5, other primers were chosen (and sometimes slightly modified) from the list of primers given in Uribe-Convers et al. (2014) [29]; these fragments span the region of the LSC harboring the two inversions which are characteristic for Asteraceae [32], but are not found in Orobanchaceae [29]. Furthermore, primer ndhI.194R (fragment 15) was replaced by the reverse-complement of primer ndhA.535F (fragment 14) due to amplification problems, and several primers were slightly modified. All used primer sequences are available in S2 Table. After these modifications, estimated fragment sizes in Artemisia varied between ~6,000 and 12,300 bp, with a minimum overlap of 173 bp among fragments, except for a small sequence gap between fragment 14 and 15 (due to the double use of primer ndhA.535F), which was filled by additional Sanger sequencing (see below). PCR reactions were performed using the Invitrogen Platinum SuperFi Green PCR Master Mix (Thermo Fisher Scientific). Amplicons were purified either using an ethanol precipitation protocol following Kück et al. (2005) [33] with slight modifications (e.g., diluting PCR products 1:5 with water before processing, and elongated incubation in ethanol, for three hours), or with the AmpliClean^™ Magnetic Bead-based PCR cleanup (Nimagen, Nijmegen, Netherlands), and were checked by gel electrophoresis before and after purification. DNA concentration and purity was again determined via Nanodrop and a Qubit 2.0 fluorometer (Thermo Fisher Scientific). All 16 fragments were then equimolarily pooled for each sample and subjected to library preparation procedures.

2.3. Illumina sequencing

Four libraries were generated for Illumina sequencing, two each for L. vulgare and L. virgatum, with different fragment sizes for two MiSeq runs of 300 bp and 80 bp paired-end reads, respectively (see below). Library preparation was done using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England BioLabs, Frankfurt, Germany) and the protocol for use with inputs of ≥ 100 ng DNA. 5 min (L. vulgare) and 10 min (L. virgatum) of enzymatic fragmentation produced fragments of around 400 bp; 20 min (L. vulgare) and 35 min (L. virgatum) of fragmentation were necessary to obtain ~200-bp fragments. As post-ligation size selection following the manufacturer’s protocol did not work, a two-step selection using AMPure XP beads was developed, where, for the 300-bp run, 0.7x beads to DNA volume would remove fragments < 400 bp in a first step, and re-suspending the eluate DNA with 0.55x beads would keep fragments of > 600 bp bound to the beads in a second step, the supernatant hence containing fragments of the desired length (here around 500 bp). For the 80-bp run, a larger fragment range was obtained using a similar approach, by first binding and discarding fragments larger than 500 bp using 0.65x beads and then concentrating the supernatant with 1.2x beads, which would bind everything except unwanted very small molecular weight DNA. The eluate thus consisted of fragments of c. 100–500 bp length, with a peak of c. 330 bp. As the PCR enrichment of adapter-ligated DNA favored smaller fragments, the final libraries had a peak length of around 300 bp for the 300-bp run and c. 220 bp for the 80-bp run. The 6-bp NEBNext indices 6 and 12 were used for barcoding L. vulgare and L. virgatum, respectively. Samples were evaluated on an Agilent 4200 TapeStation using the High Sensitivity D1000 ScreenTape assay (Agilent Technologies, Waldbronn, Germany). For the 300-bp run, the two libraries then were pooled with 48 libraries of other projects and subsequently sequenced on 0.09% (L. virgatum) and 0.08% (L. vulgare) of one lane on an Illumina MiSeq system (Illumina, San Diego, California, USA) at the Department of Biochemistry I at the University of Regensburg. The 80-bp run was conducted on the same sequencer; L. virgatum was sequenced on 1.13% and L. vulgare on 1.11% of one lane, together with 22 other libraries.

2.4. Nanopore sequencing

A Nanopore sequencing library was generated for L. vulgare using the 1D Native barcoding genomic DNA protocol, with the PCR-free SQK-LSK108 Ligation Sequencing kit and EXP-NBD103 Native Barcoding Expansion 1–12. Library preparation was done according to the manufacturer’s protocol except for some minor modifications; also, no DNA fragmentation and no FFPE DNA repair was performed. As the library contained one other sample, L. vulgare was barcoded with the 24-bp barcode NB 07. Sequencing was performed using a FLO-MIN106 flow cell on a Nanopore MinION sequencing device (Oxford Nanopore Technologies, Oxford, UK) at the Department of Microbiology at the University of Regensburg.

2.5. Complementary Sanger sequencing

For exact determination of the LSC/IR/SSC boundaries, primers were designed spanning the four junctions (S2 Table) and amplified PCR fragments were then Sanger sequenced. Likewise, primers were designed to span the gap between long-range PCR fragments 14 and 15. Sanger sequences from this gap region were used to correct and/or complete all eight assembled consensus sequences generated in the present study.

2.6. Processing of Illumina reads and genome assembly

2.7. Processing of Nanopore reads and genome assembly

2.8. Annotation of chloroplast genomes

The two complete, finished sequences from Illumina de novo assemblies of L. vulgare and L. virgatum were annotated using the online tool GeSeq [53] available at https://chlorobox.mpimp-golm.mpg.de/geseq.html. The BLAT [54] searches were done setting the protein search identity to 80% and the rRNA, tRNA and DNA search identity to 85%. As references, two closely related chloroplast genomes (from tribe Anthemideae) available in Genbank were used: Artemisia frigida and Soliva sessilis Ruiz & Pav. (subtribe Cotulinae; Genbank ID NC_034851.1). For chloroplast CDS and rRNAs, the curated MPI-MP Embryophyta chloroplast references were used as well. A HMMER profile search and tRNA annotation using ARAGORN v.1.2.38 [55] (with standard settings as on the website) were conducted additionally to check for possible annotation conflicts. After automatic annotation, putative annotation errors or discrepancies resulting from different references or algorithms were solved manually, deciding in favor of the closest references or the majority of algorithms / (published) references. The .gb file was also edited to include the very small 5'-exons of the petB, petD and rpl16 genes. The final annotated chloroplast genomes were converted into graphical maps using the OGDRAW [56] online tool [57], available at https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, with standard settings except that introns were also included in the genome map. For submission of the annotated cp genomes to Genbank, the online tool GB2sequin [58] was used.

2.9. Comparison of L. vulgare assembly results from different methods to the Illumina de novo assembly

Finalized L. vulgare chloroplast genome sequences resulting from the different sequencing and analysis pipelines (Illumina, Nanopore, mapping assembly and de novo assembly) were compared to the Illumina de novo assembly. The same was done for the L. virgatum Illumina de novo assembly and the Artemisia frigida reference. All comparisons were calculated without the IR_A. Each pair of sequences (with L. vulgare Illumina de novo at the top) was first aligned online using MAFFT v.7 [59] (available at https://mafft.cbrc.jp/alignment/server/), employing the FFT-NS-2 progressive method and otherwise standard settings. The alignment was adjusted to the length of the Illumina de novo reference, either by deleting bases from or by adding N’s to the sequence to be compared. The prepared alignments were then analyzed using the shell script alignment_info3.sh (written by Ulrich Lautenschlager, Regensburg, available upon request), which identified the total number of mismatches, substitutions, insertions and deletions between two sequences. The length and G+C content of each assembly was identified using BioEdit.

2.10. Sequence divergence and potential molecular marker identification

Apart from comparison with alignment_info3.sh as outlined above, sequence variation between the L. vulgare and L. virgatum Illumina de novo assemblies was assessed via mapping processed L. virgatum Illumina reads to the L. vulgare Illumina de novo assembly. Mapping, read deduplication and variant calling was done according to section 2.6.2.; during mapping, maxindel was set to 300 in BBMap. After packing and indexing, the resulting .vcf file was analyzed regarding variant density with VCFtools. The number of variants was output in 1,000 bp windows and graphically illustrated. Corresponding to the highest peaks in the resulting graph (windows containing more than eight variants), the most variable regions in the cp genome were identified and the respective molecular markers (intergenic spacers / genes) examined regarding total variant numbers and variation in relation to marker length.

3.1. Long-range PCR, sequencing, and genome assembly

The lengths of long-range PCR fragments obtained from Leucanthemum vulgare and L. virgatum were similar to those found in Artemisia frigida, with the shortest and longest fragments in L. vulgare being 6,086 bp (fragment 4) and 12,499 bp (fragment 14) long, respectively. Read statistics for all three runs are shown in Table 1. Sequencing of L. virgatum resulted in a total of 381,932 raw reads. 0.7% contaminant reads were removed, and length and quality filtering reduced the dataset by another 43.8%, leaving 212,186 trimmed and filtered high-quality reads (55.6%) for subsequent analyses. Sequencing of L. vulgare yielded 373,518 raw reads, of which 1.5% belonged to the PhiX spike-in and 32% were removed during length and quality filtering. Thus, 248,456 reads (66.5%) remained for analysis. Approximately 1.5 hrs sequencing of L. vulgare on a flow cell with two multiplexed samples using Oxford Nanopore MinION resulted in a total yield of 632.9 megabases (Mb) with a mean read quality of 7.49. A fraction of 22.6% from all raw reads of the run within the Albacore "pass" folder was discarded by Porechop due to middle adapters. After demultiplexing and adapter trimming by Porechop, 52,223 L. vulgare reads with a total of 161,521,432 bp were obtained, with read lengths ranging from 101–26,011 bp, an average read length of 3,093 bp and mean sequence qualities ranging from Q8—Q16 (with 48.8% of the reads having Q12 or Q13). A total of 217 reads was removed during primer trimming and quality- and length-filtering, leaving 52,006 reads of which 12,404 were used for mapping assemblies. For de novo assembly, 19,111 putative contaminant reads were additionally removed, leaving 32,895 reads. Chimera-splitting by Pacasus raised that number to 59,874 reads, of which 19,311 reads had lengths below 50 bp and were excluded. Thus, 40,563 reads were available for de novo assembly; these had an average read length lowered by about 750 bp compared to the contaminant-removed reads. For the hybrid de novo assembly, read improvement by Nanocorr lowered the read number to 38,107, but added a total of 675,084 bp to the remaining reads. All sequenced raw reads that were analyzed in the course of the present study are available at the NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA602588.

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Table 1. Read statistics for Illumina and Nanopore sequencing runs.

https://doi.org/10.1371/journal.pone.0226234.t001

For the mapping assemblies, detailed statistics are available in Table 2. Mapping Nanopore data to the Artemisia reference yielded one multiallelic site in the .vcf file, which was resolved manually by discarding the variant with the lower quality in the file. All other observed variants were biallelic. Mapped reads comprised 95.7–98.2% of the input data, with the percentage of ambiguously mapped reads being higher in Illumina (14.6% on average) than in Nanopore reads (5.97%). Mean coverage was well over 100-fold in all mapping assemblies, but with large standard deviations. However, over 99% of the reference still had coverage ≥ 30-fold for Illumina and ≥ 100-fold for Nanopore data. Variant calling resulted in 379 variants with a mean depth of 102.42-fold and a mean variant quality of 50.7 for the Illumina mapping (virgatum), 1,925 variants (depth 112.98-fold, variant quality 50.66) for the Illumina mapping (Artemisia), 297 variants (depth 685.48-fold, variant quality 16.77) for the Nanopore mapping (virgatum) and 1,645 variants (depth 721.59-fold, variant quality 16.72) for the Nanopore mapping (Artemisia).

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Table 2. Mapping statistics for reference-based assembly of Illumina and Nanopore data.

https://doi.org/10.1371/journal.pone.0226234.t002

De novo assembly of Illumina data from L. virgatum produced five contigs (see Table 3), of which three belonged to the IR; one unnecessary connection between two IR contigs had to be removed before doubling and merging. Leucanthemum vulgare data yielded four contigs, one of which (a small, unconnected contig of 1,916 bp, see Discussion section 4.6.) did not blast as chloroplast sequence and therefore was excluded. The remaining three fragments corresponded to the LSC, SSC, and IR. Fragment lengths in both species varied between 621 and 82,675 bp. Mean read coverages were 128.30-fold and 152.28-fold, respectively. In L. vulgare, only 0.26% of the assembled sequence was covered at below 30-fold while in L. virgatum, 6.62% of the sequence length had lower coverage. However, 98.66% of the assembly still was covered at least 20-fold. De novo assembly of Nanopore-only data in L. vulgare started with 8,338 reads for correction and used 4,632 reads for the actual assembly, which resulted in six contigs. One of those was discarded as it was a chimera with one of two parts being a contaminant which did not blast to Artemisia at all (see Discussion section 4.6.); another was split into two parts, resulting in six final contigs with fragment lengths between 6,655 and 53,891 bp. Polishing rewrote the contigs with 118 substitutions, 1,100 insertions and 46 deletions. Contigs were merged together with the 480-bp Sanger sequence of the primer gap (see section 2.5.). For hybrid de novo assembly with Nanocorr, 14,351 reads were used for correction and 10,422 reads for assembly; the latter yielded five contigs, of which two had to be split. The seven final contigs had lengths between 6,819 and 54,677 bp. Mean read coverage was considerably higher for the Nanopore-only assembly (321.43-fold) than for the Illumina assemblies; nevertheless, 8.83% of the assembled sequence was covered by 99 or fewer bases. However, coverage was at least 50-fold for 99.83% of the assembly. For the hybrid assembly, where there was less need for keeping coverage low (559.39-fold), the amount of bases at coverage below 100-fold accordingly dropped to 0.29%. Hybrid de novo assembly with Unicycler resulted in contigs identical to those from Illumina-only de novo assembly; results are therefore not separately shown here.

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Table 3. De novo assembly statistics.

https://doi.org/10.1371/journal.pone.0226234.t003

3.2. Genome organization, features and gene content

The Leucanthemum vulgare chloroplast genome (Fig 2) as assembled de novo from Illumina reads was 150,191 bp in length, the L. virgatum plastome was 150,120 bp long (S1 Fig); their overall G+C content was 37.45% (S3 Table). The genome sequences alongside their annotations have been deposited in NCBI’s Genbank under accession numbers MN989913 and MN996243. The two cp genomes were highly similar, featuring an LSC region of 82,675 and 82,641 bp, respectively, an SSC region of 18,400 vs. 18,435 bp, and a pair of inverted repeats of 24,558 bp in L. vulgare and 24,522 bp in L. virgatum (S3 Table). The LSC/IR/SSC boundaries of both species as determined by Sanger sequencing are depicted in Fig 3. In L. vulgare, the IR extended slightly further into the rps19 and ycf1 genes compared to L. virgatum. In the latter, the SSC region was assembled in reverse-complement direction to L. vulgare (see Discussion section). Both genomes possess the two inversions typical for Asteraceae: one large (Inv 1) and one small inversion (Inv 2) with respect to Nicotiana tabacum L. and other non-Asteroid families, located within the LSC [32].

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Fig 2. Chloroplast genome map for Leucanthemum vulgare.

Genes on the outside of the outer circle are transcribed counterclockwise, genes on the inside are transcribed clockwise. Introns are illustrated with white color within genes; genes containing an intron are additionally marked with *. Pseudogenes are preceded by a ψ. The trans-spliced rps12 gene is marked with °. Color-coding of genes depicts their affiliation to the functional groups given. The inner circle indicates the borders of the large single-copy (LSC) and small single-copy (SSC) regions as well as the inverted repeats (IR). The innermost gray shaded area shows the G+C content of the cp genome. The gene order is identical in L. virgatum (see S1 Fig), whereas their exact positions and the extent of the inverted repeat slightly differ (Fig 3).

https://doi.org/10.1371/journal.pone.0226234.g002

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Fig 3. Comparison of the inverted repeat (IR) boundaries in Leucanthemum vulgare and L. virgatum.

Genes above the green bars are transcribed in reverse direction, those below in forward direction. For both taxa, the IR extends into the ycf1 and rps19 genes, resulting in two pseudogenes (denoted by a ψ) at the single-copy / IR junctions. Lengths are given for whole genes and their duplicated fragments as well as the large single-copy (LSC), small single-copy (SSC) and IR regions (IR_A and IR_B). Arrows show basepair (bp) distance from the junctions for the ndhF and trnH genes. The SSC in L. virgatum is exemplarily reverse-complemented with respect to that in L. vulgare; both configurations exist in individual plants according to Palmer (1983) [73]. The figure is not to scale.

https://doi.org/10.1371/journal.pone.0226234.g003

The arrangement and order of genes was identical in the two species: the cp genomes contained 80 predicted protein-coding genes, four rRNA genes, and 30 tRNA genes coding for all 20 amino acids. Seven protein-coding genes, all rRNA genes and seven tRNA genes were duplicated due to the IR, raising the total number of genes to 132. Six of the 30 tRNA genes and 12 of the 80 protein-coding genes contained introns; of the latter, two harbored two introns while the rest had one single intron. Two pseudogenes were present in each genome: ψ-ycf1 and ψ-rps19 were located at the IR/SC boundaries (Fig 3), their lack of functionality being due to only partial duplication. The trans-spliced rps12 gene was found in the LSC (5'-end) and the IR regions (duplicated 3'-end). A summary of all genes is given in Table 4.

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Table 4. Genes present in the sequenced Leucanthemum vulgare and L. virgatum genomes.

https://doi.org/10.1371/journal.pone.0226234.t004

3.3. L. vulgare and L. virgatum sequence divergence and molecular marker identification

The L. virgatum cp genome featured 99.17% nucleotide identity to L. vulgare (see S3 Table), the cp genome of Artemisia frigida was 96.31% identical. Alignment of both genomes yielded a total of 4,704 mismatches, while L. virgatum differed from L. vulgare in only 1,038 mismatches, 28.8% of which were substitutions, 37.7% were gaps, and 33.5% were inserted bases. Analysis of the variant density of L. virgatum with respect to L. vulgare showed six regions of high variance (Fig 4), corresponding to three intergenic spacers and two genes (ndhF and ycf1), which varied in 8–28 positions and had a variability of 0.55–1.38% (Table 5). The most promising intergenic spacer was trnE-rpoB, with 12 variants within an easily amplifiable length of 869 bp. The ycf1 gene featured two variability hotspots of 794 bp (1.39% variability) and 488 bp (1.84% variability), respectively; at the end of the ndhF gene, one hotspot of 750 bp with 1.6% variability was found.

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Fig 4. Nucleotide variant density in L. vulgare and L. virgatum chloroplast genomes.

Illumina reads from L. virgatum were mapped to the Illumina de novo assembled sequence of L. vulgare (lacking the second inverted repeat) and variants called. Plot data on variant density generated by VCFtools in 1000-bp windows. The position of the first inverted repeat (IR_B) is indicated by red lines. Peaks depicting potentially useful marker candidates for Leucanthemum are highlighted by arrows.

https://doi.org/10.1371/journal.pone.0226234.g004

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Table 5. Chloroplast molecular markers with high variability between L. vulgare and L. virgatum.

https://doi.org/10.1371/journal.pone.0226234.t005

3.4. De novo and mapping assemblies of L. vulgare with Illumina and Nanopore data

The seven assembly methods tested on L. vulgare resulted in cp genome sequences of lengths ranging between 125,633 bp (100%) in the Illumina de novo assembly (and the hybrid de novo assembly using Unicycler, not shown) and 124,851 bp in the Illumina mapping (Artemisia) assembly (Table 6). The sequences (except from the Illumina de novo assembly which was deposited in Genbank, see above) are available in S2 File. Setting Illumina de novo as the gold standard, the Nanopore hybrid assembly using Nanocorr achieved the highest identity with the former (99.98%). Most divergent from the standard was the Nanopore mapping (Artemisia) with 98.29%. The quality of the Nanopore mapping approach increased when using the more closely related reference L. virgatum (99.51%), which is almost equal to the Nanopore de novo assembly (99.59%) and better than mapping the high-quality Illumina data to the more distantly related Artemisia (99.27%). Generally, de novo assembly always yielded better results than mapping assembly. Unlike in the comparison between the two Leucanthemum species, where substitutions, gaps, and inserted bases contributed more or less equally to the total number of mismatches (S3 Table), deletions were the main source of error in all tested approaches, accounting for 56.2–96.0% of the differences. Nanopore mapping seemed to yield the highest proportion of insertion errors (32.1–34.6%), while, compared to the other approaches, most erroneous substitutions were introduced by Nanopore de novo assembly (18.5%).

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Table 6. Comparison of six assemblies of the Leucanthemum vulgare plastome, obtained using different methods and data types, with a "gold-standard" Illumina de novo assembly.

https://doi.org/10.1371/journal.pone.0226234.t006

4.1. Enrichment of chloroplast DNA using long-range PCR

For the present study, we relied on enrichment of cp DNA via long-range PCR. For this approach, several primer sets are available (e.g., [60], producing nine fragments). The protocol chosen here [29] resulted in 16 fragments with a maximum length of 12,499 bp. A limitation of the approach is that highly degraded DNA cannot be used for long-range PCR, which limits its use with herbarium material [6,61]. The method is also unsuitable if larger rearrangements in gene order are to be expected, as have been found for example in Passiflora L. [62]. In any case, optimization of primers for the study group will almost always be necessary to amplify all fragments (in the present study, different primers had to be used for five out of 16 fragments, and several primers were slightly modified). It is also essential to use a high-quality-, proofreading polymerase to avoid misinterpretation of erroneously incorporated bases as true variants, and to remove unwanted primer sequence during read processing.

The main disadvantage of PCR-based enrichment however seems to be the resulting uneven read coverage, which is partly due to doubled sequencing at PCR-fragment overlaps. However, apart from that, although fragments were pooled at equimolar ratios before sequencing in the present study, fragment coverage across the cp genome turned out to be very variable for Illumina as well as Nanopore reads (Fig 5). Possible reasons for such a behavior include adapter ligation biases and differences among targets regarding denaturation and annealing during library preparation [2]. The same effect was mentioned by Doorduin et al. (2011) [63], who by contrast found much lower variation in coverage when using DNA from isolated chloroplasts. Altogether, long-range PCR thus cannot be regarded the optimal approach for cp genome sequencing, also because exceptionally high coverage in only a subset of regions might cause problems during Nanopore de novo assembly (see section 4.6.). A suitable alternative might be "genome skimming" [6,64] or solution-based hybridization ("target enrichment") via custom-designed oligonucleotide probes representing the cp genome [65,66]. However, target enrichment requires a closely related reference for bait design, and genome skimming might require extensive sequencing to obtain sufficient chloroplast read coverage in plants with large nuclear genomes. Chloroplast DNA can also be obtained by isolating whole chloroplasts before sequencing. However, a high amount of fresh leaves is required for isolation [6], which may exceed 5 g or even 20 g [67].

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Fig 5. Chloroplast genome read coverage of the Nanopore de novo assembly.

Leucanthemum vulgare Nanopore long reads used for assembly (post-correction and post-trimming) were mapped to the assembled sequence lacking the second inverted repeat (IR_A) and coverage-across-reference plot data extracted with Qualimap. Black lines represent the long-range PCR fragments reads are based on. Note the uneven coverage across fragments and coverage peaks at fragment overlaps. bp, basepairs.

https://doi.org/10.1371/journal.pone.0226234.g005

4.2. Assembly of chloroplast genomes using NGS

The assembly of cp genomes using Illumina short reads is still regarded as the "gold standard" regarding data quality and integrity. The most common outcome of de novo plastid assembly today are three long contigs corresponding to the LSC, SSC, and IR regions [6], as presented for example in Wang et al. (2018) [21] and the present study. The G+C content of the two Illumina de novo assemblies presented here is typical for cp genomes [68] and suggests that no large portions of nuclear DNA have been erroneously incorporated. Both plastomes are highly similar (0.83% sequence divergence); as either species belongs to one of two main clades found in Leucanthemum [69,70], this possibly represents the average cp genome divergence within the genus. The cp genome of Artemisia frigida, belonging to a different subtribe, still is only 3.69% divergent from L. vulgare, which points toward close relationships within Anthemideae as a whole. The SSC region of L. virgatum was assembled to be reverse-complemented with regard to that in L. vulgare. While such differences have been highlighted as "unique sequence rearrangement event" by Liu et al. (2013) [71] and also in other studies (see [72]), it has already been shown by Palmer (1983) [73] that molecules of both SSC orientations exist equimolarily within the same individual. It is also important to realize that with the use of PCR fragments that do not span the entire IR, it is per se impossible to distinguish between the two orientations.

The comparison of the two new cp genomes with regard to sequence variability yielded five molecular marker candidates with potential for use in phylogenetic studies in Leucantheminae / Anthemideae. The intergenic spacers trnE-rpoB, ndhF-rpl32 and rpl32-trnL were already described in other studies as being phylogenetically informative within angiosperms or specifically within Asteraceae [74,75]. The ycf1 gene has been recognized as an interesting marker for barcoding, since it contains a high percentage of parsimony-informative characters, which are mostly due to the frequent occurrence of SSRs in the region [76,77]. Another interesting aspect is the high variability at the end of the ndhF gene, in the region close to the IR/SSC boundary. However, sequencing of the entire ycf1 or ndhF gene will not be too effective in terms of variability per sequenced length, so if their use as a molecular marker is intended, it would be advisable to design suitable primers around their variability hotspots to reduce Sanger sequencing costs. The generally lower substitution rate of the IR region, which has been demonstrated in several plant groups (see [78]), is attributed to its duplicative nature in combination with the effects of biased gene conversion [79,80].

4.3. Hybrid assembly approaches

Three basic procedures can be used for hybrid genome assembly: either, an assembly is first generated based on Nanopore data alone and is subsequently polished with Illumina data (e.g., in [81]), often by using the tool Pilon. Alternatively, the recently published pipeline Unicycler follows a short-read-first approach, by assembling short reads to an assembly graph which is subsequently resolved using long reads. Third, long reads can be corrected by aligning short reads prior to assembly, for the benefit of providing high-quality input to the latter (e.g. by Nanocorr).

In the comparison of hybrid and non-hybrid approaches presented by Wang et al. (2018), the authors found that the hybrid approach performed best, yielding a single high-quality (final error rate: 0.0007 per base of a set of validation reads) contig representing the whole cp genome, as long as 20-fold coverage of long and short reads was provided [21]. Hybrid assembly was also superior to Nanopore-only assembly in [82], where the authors sequenced a mitogenome from Chrysanthemum L. The same effect was observed in the present study. Hybrid assembly using Unicycler (results not shown) yielded results identical to de novo assembly of short reads alone (three contigs), but was not able to assemble a continuous contig covering the two IRs. This came as no surprise as the longest Nanopore read was too short with only 12,532 bp, matching the length of the longest PCR fragment sequenced. The use of long-range PCR thus limits the possibilities of Unicycler and Nanopore reads in general. Wang et al. (2018) [21] also noted that hybrid assembly of a single contig was already possible with a coverage of 5-fold of Nanopore reads of lengths > 30 kb (together with at least 8-fold short-read coverage), but failed when Nanopore read lengths were < 20 kb long (i.e., shorter than the IR region). Another hybrid approach pursued in the present study (correction of long reads using Nanocorr before assembly) resulted in five contigs of which two had to be split, and the final sequence was not completely identical to the Illumina de novo (and thus, the Unicycler hybrid) assembly (99.98%). This means that assembly based on short reads but supplemented with information from long reads produced better results than proceeding the other way around.

4.4. De novo assembly based on Nanopore data alone

Only very few studies have applied Nanopore sequencing exclusively for cp genome assembly. Bethune et al. (2019) used Nanopore combined with target enrichment to sequence seven taxa from Poaceae and Arecaceae, of which one was subsequently used as reference for the others [24]. From the six assemblies which were conducted with the Flye assembler [83], none recovered the plastomes in a single contig, but two or more contigs of varying length. The authors [24] specifically noted the influence of input DNA quality on assemblies. DNA isolated from fresh tissue resulted in longer median read lengths and only two contigs with higher plastome coverage (with respect to the reference) than silica gel-dried samples. As mentioned above, this again underscores the importance of read length for Nanopore assembly. Wang et al. (2018), in their comparative study mentioned above, found that Canu was able to assemble the Eucalyptus pauciflora Sieber ex Spreng. cp genome into one or two contigs depending on read coverage, which however lacked some genome regions or covered others twice [21]. No assembly covered the complete cp genome; the final error rate after polishing with Nanopolish decreased to 0.0022 (compared to 0.0007 in the hybrid assemblies). Our Nanopore-only assembly yielded slightly better results: the Canu assembly produced six contigs, which however covered all of the cp genome, except the first 152 bp. Although overlap among the contigs was up to ~830 bp, no regions were covered twice. As a side note it is important to realize that the often advocated advantage of de novo assembly, namely no need for a reference genome, is not completely met for cases in which more than one contig is assembled. In our analyses, the six contigs all overlapped, but with very variable overlap lengths, one overlap being as little as seven bp, five of which were a C-homopolymer. In such cases, reliable merging of contigs is often realized by mapping to a reference sequence (e.g., [84]; see also [85]), as also done here.

Although the Nanopore-only de novo assembly presented in our study could not fully keep up with that from the hybrid approach using Nanocorr (sequence identity to Illumina de novo 99.98%, see Table 6), it reached a comparatively high sequence identity of 99.59%. This is comparable to the final 99.5% nucleotide identity of the assembly by Loman et al. (2015) [11], whose methods were also applied in the present study (read correction with a POA graphs—dependent consensus algorithm as implemented in Canu, polishing of the final assembly with Nanopolish). Regarding the remaining errors in our assembly, almost 70% of all mismatches were gaps; the accumulation in deletion errors in homopolymer runs is clearly visible in visualizations of mappings prepared for the mapping approach and increases with homopolymer length. This fits the expectation for non-random Nanopore sequencing errors mentioned in Laehnemann et al. (2016) [7].

4.5. Reference-based assembly using Nanopore data

Apart from de novo assembly, cp genome sequences can also be obtained by simply aligning reads to a reference genome and creating a consensus sequence from the mapping. This approach is only suitable for studies of closely related taxa with uniform chloroplast genomes [2,6]. The reason for that is that a mapping consensus will always adopt characteristics of the reference from which it was inferred. Structural rearrangements or additional transferred genes might easily be missed, especially when mapping short reads; the structure of repeat regions cannot be distinguished by mapping based approaches at all [86]. For the assembly of the globe artichoke (Cynara L.) cp genome with Illumina reads, Curci et al. (2015) [76] compared mapping and de novo approaches and found the results to be almost identical. Sancho et al. (2018) [87] analyzed intraspecific diversity in Brachypodium P.Beauv. with Illumina data. According to their results, the reference-guided approach yielded fewer and longer contigs than de novo assemblies in most cases. In the present study, mapping assemblies based on Illumina data were found to be of somewhat lower quality than those from de novo assembly, being 99.27% and 99.74% identical to the latter, respectively, depending on the mapping reference used.

However, to our knowledge, no study has yet employed Nanopore sequencing reads for reference-based assembly of plant chloroplast genomes. The mapping assemblies of L. vulgare presented here were produced using read lengths of up to 3,000 bp and two closely-related references, which do not show any structural reorganization compared to L. vulgare and also have the same gene order (nucleotide identities to L. vulgare Illumina de novo: L. virgatum 99.17%, A. frigida 96.31%). Illumina mappings were superior to Nanopore mappings for both references, but surprisingly, nucleotide divergence (from the Illumina de novo assembly) was only slightly worse in Nanopore mappings, with the difference between the two being as low as 0.23% when using the L. virgatum reference. However, this difference rose to 0.98% when using Artemisia. This loss in quality caused by relying upon a more distantly related reference was found for both datatypes, but seemed to be worse with Nanopore: Illumina assemblies suffered a quality decline of 0.47% nucleotide identity when switching to the more distant reference, Nanopore assemblies declined by 1.22%. This emphasizes the great importance of choosing a suitable reference in mapping-based assemblies of Nanopore data. Using the L. virgatum reference produced a consensus of almost comparable quality to the de novo Nanopore assembly, and superior to the Illumina assembly based on the more distant reference (see Table 6).

A second essential point for obtaining high-quality reference-based assemblies with Nanopore as well as Illumina data is the choice of software used for mapping and variant calling. Preliminary tests with popular short-read mappers (Bowtie2 [88], BWA-MEM [89], and BBMap) showed that despite all three tools claim to support gapped alignment, only BBMap (which also supports long reads) was able to correctly map reads spanning large gaps (for mapping against the Artemisia reference, the largest deletion occurring in L. vulgare had a length of 459 bp). Bowtie2 and BWA-MEM either soft-clipped the respective reads or did not map them at all, leaving the site of deletion with a coverage gap. However, correct mapping across deletions is of utter importance as most variant callers will call the reference sequence at positions where read information is lacking, which would distort the resulting consensus sequence. Tested consensus-calling pipelines also included BCFtools mpileup plus call for Illumina data, which however was not able to correctly call indels as variants. For Nanopore data, variant calling was only successful using callvariants2.sh after mapPacBio.sh mapping.

An advantage of reference-based assembly is that the assembly process can be easily monitored by comparing the visualized mapping (e.g., in IGV) to the final called consensus. Specific patterns produced by the used datatype or algorithm can thus be taken into account and potential errors avoided. For example, when mapping Nanopore reads, especially insertions will often not be mapped at a precise location, but slightly shifted up- or downstream in several reads, thus blurring the signal for callvariants2.sh, which will ultimately call the reference in these cases. This problem is aggravated with more distant references as exact alignment gets more difficult. Regarding the variant-calling process, mismatches and small (1-bp or 2-bp) indels were usually captured correctly in both Illumina and Nanopore mappings. By contrast, larger insertions in L. vulgare with respect to the reference were more likely to be ignored, leading to the large number of deletions observed in the consensus sequences (56.2–89.4% of all mismatches, see Table 6). This effect seems to be due to the used algorithm, intensifies with insertion length as well as more distant references, and is generally more pronounced with Nanopore data due to blurred mapping as described above. Deletions in the mapped taxon with respect to the reference were however more often called correctly from Nanopore reads, and almost always from Illumina reads. Callvariants2.sh also deals well with declining read coverage in homopolymer runs and mostly makes the correct full-length call.

4.6. Optimizing Nanopore de novo and reference-based assembly

A read coverage of at least 30-fold is generally recommended for Illumina de novo as well as reference-based plastome assembly [6,64]. In our Illumina de novo assembly of L. vulgare, only 0.26% of bases received coverage below that threshold (Table 3). Accordingly, the Unicycler pipeline yielded the lowest possible number of contigs when using short reads and no read extension methodology [6]. The L. virgatum assembly, with 6.62% of bases having fewer than 30-fold coverage, resulted in five contigs. Although a complete cp genome sequence was easily obtained, this somehow suboptimal result might be due to a lowered k-mer frequency as a consequence of low coverage in certain regions. Regarding Nanopore de novo assembly, a coverage of 30-fold will likely not yield optimal results (but see [11]). However, a minimum of 50-fold coverage in over 99.5% of the bases seemed to be sufficient for reliable assembly (Table 3). If coverage is too low, problems might arise with reads derived from PCR fragments with only small initial overlap; after adapter-, primer-, and quality trimming, too many reads might get too short, precluding a continuous assembly. Fragment 9 and 10 in our analyses had an overlap of only 134 bp between the two primer sequences; this resulted in a very low coverage of down to 5-fold in the Nanopore-only assembly, and even a gap in a preliminary run with different settings (not shown). In Nanopore mapping assemblies, very low coverage might result in variants not being called, which will compromise the resulting consensus.

However, a much more important problem observed with Nanopore data is the detrimental effect of too high coverage. Wang et al. (2018) recognized an effect of too low or high coverage on assembly quality, stating that "there is a complex relationship between assembly accuracy and input read coverage when assembling chloroplast genomes with ONT data using Canu" [21]. Twyford and Ness (2017) [6] mention that de novo assembly performance could be improved by downsampling the input reads, and Izan et al. (2017) [86] stress that very high coverage may result in alternative assemblies which can lead to contig fragmentation. For the present study, preliminary de novo assembly tests (results not shown) with a reduced compared to the full dataset considerably decreased the number of contigs (approximately by half). The observed effect is likely due to the fact that at very high coverage, erroneous variants introduced by PCR or sequencing errors will be frequent enough in a dataset to pass the threshold for being incorporated into (extra) contigs by the assembler. To avoid this, a coverage-reduced dataset should be used for de novo assembly, for example via the readSamplingBias parameter in Canu. This has the side-effect of preferentially using the longest reads for analysis, which might also help to improve the results. With long-range PCR based reads, however, care must be taken to ensure that reads originating from short fragments are not accidentally filtered completely.

Filtering possible nuclear or contaminant reads from plastome reads, for example by mapping to a known reference, is a common procedure prior to assembly. However, applying too stringent filters might result in the loss of true plastid reads in the case of atypical cp genomes or such which have incorporated mitochondrial or nuclear DNA [6]. For this reason, read filtering other than removing the PhiX spike-in was omitted in Illumina de novo analyses shown here. Unicycler proved relatively robust to this kind of perturbation: apparently, present contamination reads in the L. vulgare sample were assembled into an additional, isolated contig (showing BLAST matches in Proteobacteria) that could simply be excluded from further processing. In contrast to Unicycler, Canu was much more susceptible to contaminant-related errors. Even though input reads were subjected to BLAST filtering prior to de novo assembly, the latter still yielded a chimeric contig with one large part matching the Artemisia chloroplast and a small part blasting within Proteobacteria. Although the removal of this contig did not deteriorate the quality of the final assembly, the risk of obtaining erroneous contigs might be higher with Canu.

Another severe problem with the Nanopore reads used here for de novo assembly was the occurrence of chimeric reads. Two types of chimeras were observed. The first comprised reads with a first part being identical to a second part, but in reverse-complement orientation (so-called palindromes). It remains unclear what the source of this kind of error was. Warris et al. (2018) [47] describe a similar phenomenon in long reads that can occur during whole-genome amplification, so palindrome formation in cycles of long-range PCR at least seems conceivable. In contrast to the short, fragmented Illumina reads, the long Nanopore reads will introduce these errors into assembly. To avoid this, before starting de novo assembly we subjected reads to Pacasus, which splits reads containing palindromic sequences. Splitting proved essential, as initial assemblies with unsplit reads produced a large number of (sometimes palindromic) contigs, rendering impossible the generation of a proper consensus sequence.

According to White et al. (2017) [90], who analyzed several types of chimeric reads originating from MinION sequencing, these can also consist of dissimilar fragments which are concatenated by the same sequencing adapter accidentally ligated in between them during library preparation. This type of chimera might also result from a very rare, Nanopore-specific process which has been called "in silico chimerism" by White et al. (2017) [90]. Here, too fast pore reloading results in the recognition of two fragments as one by the base-calling algorithm. A corresponding filtering step is incorporated in the Nanopore bioinformatic pipeline (Porechop). In the analyses presented here, 22.6% of the total flow cell reads were discarded due to putative middle adapters. If this filtering is not set to be stringent enough, it seems possible that chimeric reads could pass the filter, and could act as bridges to connect sequence regions in Canu contigs which are actually distant from each other on the cp genome. It must be stressed here that without a reference plastome, detection of chimeric contigs is impossible; however, this technique of course harbors the risk of missing true rearrangements in the cp genome to be assembled.

As a concluding remark, the frequency of chimeras in our dataset seems to be unusually high, considering the fact that all types of chimeras found by White et al. (2017) [90] together comprised only 1.7% of the reads and Warris et al. (2018) [47] described palindromic reads as occurring during whole-genome amplification, not long-range PCR. Further, unknown processes may thus contribute to produce the phenomena observed. At least, both contaminant as well as chimeric reads are negligible problems for reference-based assemblies, as the initial mapping step will result in dismissal of the former as unmapped, while the latter will likely result in supplementary mappings, which are also excluded from the output if BBMap is used with the settings described here.

4.7. Conclusions—Lessons learned

Based on the results obtained in this study, we provide some rough guidelines on the sequencing of plant cp genomes. Regarding data production, PCR-mediated genome reduction has the advantage of being easy to realize in an ordinary lab, but will depend on the performance of universal primer sets in the studied group. Several shortcomings must also be taken into account (see section 4.1.). For enrichment of plastid DNA with subsequent Nanopore sequencing, the use of PCR must be regarded as suboptimal as it nullifies some of the main advantages of the latter. While Nanopore sequencing can now produce reads with lengths of more than two Mb [91], PCR-derived reads will currently have a maximum length of 23 kb [60]. This also precludes the disambiguation of the two IRs, which may otherwise be possible. If the use of long-range PCR plus Nanopore sequencing is intended, it is advisable to

• use a high-quality, proofreading polymerase for minimal entry of PCR artifacts into reads;
• choose fragment sizes as large as possible; this also requires the use of appropriate DNA extraction techniques;
• avoid too short overlaps among PCR fragments (preferably, > 300 bp) as in combination with low Nanopore read coverage, these might lead to fragmented assemblies;
• downsample regions of uneven read coverage, for example at fragment overlaps with doubled coverage, using VariantBam [92] or related tools; and
• account for palindromic sequences which might occur whenever PCR is used; these must be properly handled prior to assembly with the Pacasus tool or other appropriate software.

Hybrid assembly approaches with both Nanopore and Illumina data still yield better results than only using Nanopore reads. However, depending on the research question, Nanopore sequencing is also capable of delivering high-quality results at a comparatively lower cost. Our results show that the vast majority of the 221 deletion event errors within the Nanopore de novo assembly (~80%) lies within homopolymer regions of five or more bases; the same is true for almost 62% of the insertion event errors. Exclusion of such regions will greatly reduce the error rate of the respective assembly. If phylogenetic analysis of several cp genomes in a plant group is intended, the genomes can be easily analyzed after masking the respective homopolymer regions, which should be excluded from this type of analyses anyway. Of course, the degree of relatedness in the respective plant group must be considered. While in the Nanopore de novo assembly, the error rate of 0.41% sequence identity (including all errors) is much lower than the average divergence between A. frigida and L. vulgare (3.69%), the situation looks somewhat different in a close-knit species group like the genus Leucanthemum (L. vulgare—L. virgatum: 0.83%). Furthermore, when starting research on a new plant group, it will be advisable to use parallel Illumina sequencing in the first cp genome for comparison purposes.

To ensure that high-quality de novo assemblies can be obtained from analyzing Nanopore data alone, several important points should be taken into account:

• reads with middle adapters (i.e., chimeras consisting of two different reads) must be rigorously removed or at least bioinformatically split, to avoid the introduction of wrong proximity information into assembly
• for de novo assembly using Canu, the elimination of contaminant reads from other organisms prior to assembly is vital. This can be done by blasting against a curated database of known plant genomes
• read coverage should be kept at reasonable heights, especially when using uncorrected reads (as a gross starting point, not much higher than 300-fold for the final unitigging step), for example by subsampling reads before assembly (e.g., with SAMtools or BBTools) or by applying the Canu parameters readSamplingBias / readSamplingCoverage
• preferentially including the longest reads might improve assembly quality; use the above Canu parameters or the minReadLength parameter

In some situations, for example with limited computational resources or large amounts of samples to assemble, reference-based assembly might be favored over de novo approaches. However, it should not be conducted in groups with known cp genome variability or structural reorganization, but rather for population genomics or in genera of very closely related species. If these requirements are met, mapping assemblies can yield a consensus comparable to that of de novo assembly, as shown in the present study. For this, it is essential to

• use suitable software, for example BBMap plus the callvariants2.sh script. However, discrimination of the two IR copies will not be possible with BBMap as it cannot handle very long reads;
• correctly set software parameters. This may require some optimization; for example, variant calling based on a majority vote for alleles might not work for the error-prone Nanopore reads. The minimum allele fraction required for a call could therefore be lowered (e.g., to 0.45); and
• choose the closest reference available (from the same genus, ideally the same species). According to our results, the quality of Nanopore reference-based assemblies decreases disproportionally fast with more divergent reference genomes, even if these still have sequence identities above 95% to the studied taxon. The reference should include the complete IR_A or only parts of it, depending on read length, to allow unambiguous mapping of all reads.