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Article

Structural Refinement by Direct Mapping Reveals Assembly Inconsistencies near Hi-C Junctions

by
Luca Marcolungo
1,†,
Leonardo Vincenzi
1,†,
Matteo Ballottari
1,
Michela Cecchin
1,
Emanuela Cosentino
2,
Thomas Mignani
1,
Antonina Limongi
1,
Irene Ferraris
1,
Matteo Orlandi
1,
Marzia Rossato
1,2 and
Massimo Delledonne
1,2,*
1
Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy
2
Genartis srl, Via IV Novembre 24, 37126 Verona, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2023, 12(2), 320; https://doi.org/10.3390/plants12020320
Submission received: 30 November 2022 / Revised: 22 December 2022 / Accepted: 6 January 2023 / Published: 10 January 2023
(This article belongs to the Special Issue Assembly and Phasing of Complex Genomes in Plant)
Browse Figures
Versions Notes

Abstract

:
High-throughput chromosome conformation capture (Hi-C) is widely used for scaffolding in de novo assembly because it produces highly contiguous genomes, but its indirect statistical approach can introduce connection errors. We employed optical mapping (Bionano Genomics) as an orthogonal scaffolding technology to assess the structural solidity of Hi-C reconstructed scaffolds. Optical maps were used to assess the correctness of five de novo genome assemblies based on long-read sequencing for contig generation and Hi-C for scaffolding. Hundreds of inconsistencies were found between the reconstructions generated using the Hi-C and optical mapping approaches. Manual inspection, exploiting raw long-read sequencing data and optical maps, confirmed that several of these conflicts were derived from Hi-C joining errors. Such misjoins were widespread, involved the connection of both small and large contigs, and even overlapped annotated genes. We conclude that the integration of optical mapping data after, not before, Hi-C-based scaffolding, improves the quality of the assembly and limits reconstruction errors by highlighting misjoins that can then be subjected to further investigation.

1. Introduction

Accurate genome assemblies are required for robust downstream genomic analysis, including applications such as variant calling, differential expression profiling and pangenome analysis. Several approaches have been developed and benchmarked to verify and improve the accuracy of de novo genome assemblies at the nucleotide level [1,2,3,4]. However, structural accuracy is equally important, but it is much more laborious and complex to validate.
Advances in genome sequencing technologies and assembly algorithms currently allow the reconstruction of the full, gapless sequence of entire chromosomes [5] and even entire genomes [6,7], as demonstrated by the recent completion of the full human genome [8]. Most workflows rely on long-read technologies to generate contigs, but even the longest reads are usually insufficient to reconstruct full chromosomes. To achieve chromosome-scale assemblies, contigs must be connected by scaffolding technologies–principally high-throughput chromosome conformation capture (Hi-C) and/or optical genome mapping.
Hi-C captures genome-wide chromatin interactions in the nucleus and quantifies the interaction frequency of different loci. It involves the preparation of a chromosome conformation capture library followed by short-read sequencing. Given the assumption that intra-chromosomal interactions are more frequent than inter-chromosomal ones and that intra-chromosomal interaction frequency is directly related to the two-dimensional distance along the DNA sequence, Hi-C can statistically determine the physical proximity between genomic regions. This can be exploited for genome scaffolding by ordering and orienting contigs according to the likelihood that two contigs are in physical proximity on the same chromosome. Because intra-chromosomal interactions can span the entire chromosome, this technique enables genome reconstruction at the chromosome level [9]. In contrast, optical mapping involves the direct imaging of DNA molecules. Ultra-high-molecular-weight (UHMW) DNA is labelled at nicking-enzyme recognition sites, and long stretches (>100 kb) of DNA flowing through nanochannels are then scanned and digitalised, recording the position of labels in the DNA sequence. Such molecules can be assembled using an overlap layout consensus algorithm to generate a physical map suitable for the alignment of contigs. This procedure, known as hybrid scaffolding, usually requires contigs >100 kb derived from next-generation sequencing (NGS) data and involves two steps: the identification and correction of incongruences between maps and NGS sequences followed by the anchoring of corrected sequences to the maps, thus generating scaffolds.
In de novo genome assembly workflows, Hi-C is either used alone or applied to existing hybrid scaffolds because it can capture more distant interactions, enabling the reconstruction of whole chromosomes [10]. For example, the Vertebrate Genome Project applies Hi-C to existing optical maps, whereas the European Reference Genome Atlas relies solely on Hi-C scaffolding technology [11]. Hi-C also benefits from more straightforward laboratory protocols than optical mapping, and several companies already distribute commercial Hi-C kits. In contrast, optical mapping is dependent on laborious sample preparation and handling procedures, and it is not always possible to extract the required UHMW DNA. Furthermore, Hi-C libraries can be analysed using standard short-read sequencers, whereas a dedicated instrument is required to acquire optical maps.
Despite the practical advantages of Hi-C, its probabilistic approach can lead to scaffolding errors such as contig misplacement and misorientation [12,13,14,15,16], especially with short contigs and scaffolds [10,12]. For example, Hi-C scaffolding led to 21 misplacement and misorientation errors (affecting 83 scaffolds) in the goat (Capra hircus) genome assembly [15], as well as hundreds of such errors in chromosome-scale de novo assemblies of the human, mouse and Drosophila melanogaster genomes [14]. The use of Hi-C for scaffolding without manual revision, therefore, creates a demand for de novo assembly validation. Because optical mapping is an orthogonal technique that relies on direct DNA imaging, it has also been used to detect and correct structural errors within NGS-derived genome assemblies [17,18]. For example, optical mapping improved the structural accuracy of the mouse reference genome and a complex region in the human genome (1q21.1–q21) that was integrated into the GRCh38 reference assembly [19]. However, despite its advantages, few studies have used optical mapping data as a validation tool of the assembly [20,21,22,23], and optical maps have not been used thus far to validate or correct Hi-C-based scaffolds.
Here we used optical mapping to assess Hi-C de novo genome reconstructions. Initially, we applied the optical mapping approach to an in-house genome assembly before extending it to four other publicly available genomes. Validation with optical maps identified and corrected many Hi-C scaffolding errors suggesting a manual revision of conflicting regions, some of which comprise coding sequences. These results will serve as a guideline for genome assembly projects and possibly for the revision of completed genomes in order to maximise the accuracy of structural genome reconstructions.

2. Results

2.1. De Novo Assembly and Structural Validation of the HAEMATOCOCCUS Lacustris Genome

We generated long-read sequences of H. lacustris using Oxford nanopore Technology (ONT), producing 12 Gb of data comprising two million reads with an N50 value of 15 kb (Table S1). The reads were assembled de novo using Flye, yielding an assembly of 272 Mb with an N50 value of 250 kb (Table S2). The ONT assembly was polished using ONT, and Illumina reads to refine residual sequence errors. BUSCO analysis identified 1476 complete genes, indicating good completeness (97.1%) but a high duplication frequency of 72.7%. A subsequent purging step reduced the BUSCO duplication frequency to 10.6% while maintaining high completeness (92.6%). The assembly size was reduced to 150 Mbp (Table 1). A comprehensive description of the H. lacustris genome is being reported elsewhere [24].
An initial scaffolding step was carried out by Hi-C to order and orient contigs. A Hi-C library prepared from H. lacustris nuclei was sequenced using Illumina and generating 90 Gb of sequence data. This was integrated with the long-read data to generate a highly contiguous, chromosome-scale assembly. Specifically, the scaffolding process joined 930 contigs of the ONT assembly (91% of the assembled sequence) and generated a “Hi-C-scaffolded genome” comprising 32 scaffolds with an N50 value of 4 Mb (Table 1).
For the structural validation of the H. lacustris genome assembly based on the complete ONT dataset and the Hi-C scaffold, we produced two independent optical genome maps using the enzymes BspQI and BssSI, generating 198 and 163 Gb of sequencing data, respectively. The sizes of the resulting assembled genome maps were 323 and 370 Mb, with N50 values of 520 and 630 kb, respectively. The size of the reconstructed genome maps highlights how both haplotypes were fully reconstructed using optical maps. The hybrid scaffolding generated a genome assembly of 175 Mb with a scaffold N50 of 655 kb. This revealed a large number of inconsistencies between the Hi-C scaffold and optical maps, which fragmented the Hi-C scaffolded genome and reduced the N50 value (Table 1). A total of 864 incongruences were found, 455 of which were located near the 296 gaps introduced by Hi-C and classified as scaffolding errors by manual inspection. The number of conflicts solely arising from Hi-C incorrect gap size estimation was 14. Figure 1A shows an incongruence in which optical maps and NGS scaffolds (shown as in silico digested maps) align correctly until the mismatch point. Downstream, the labelling differs between the two datasets, highlighting a genomic region with a different structure in the two maps. The correctness of optical map reconstruction was confirmed by showing that two independent maps based on different enzymes uncovered the same incongruence. Furthermore, numerous Bionano molecules supported the reconstruction at the conflicting region and confirmed the incorrect assembly generated by Hi-C scaffolding (Figure 1B).
We then used ONT data to manually verify 62 gaps introduced by Hi-C located close to the identified conflicts. In 81% (50/62) of the revised cases (Table 2), ONT data did not support the HiC-based reconstruction because no reads spanned and properly aligned across the gap, thus confirming that Hi-C-based scaffolding introduced an error at that point (Figure 2A). In the remaining 19% of revised cases, ONT reads confirmed the contig-to-contig junction (Figure 2B). However, only a fraction of these junctions (8%) showed a good number of ONT reads in support, while <5 reads confirmed the contig connection in the remaining 11% of cases.
Hi-C-derived misjoins have been reported as more frequent for short contigs, so we evaluated the size of the contigs joined erroneously in order to establish a correlation between contig length and error probability (Table S3). Surprisingly, we found that errors did not vary significantly in relation to contig length and that the highest proportion of errors involved contigs with a consistent length of 600–650 kb. Within this range, 83% of contigs (five in every six) were involved in erroneous joins.

2.2. Structural Validation of Four Published Genome Assemblies

To determine whether the above findings were unique to our in-house H. lacustris genome assembly or could be generalised, we evaluated the presence of OM/Hi-C inconsistencies in four assemblies based on Hi-C in which the NGS-based and optical map assemblies were accessible.
First, we analysed two published assemblies of white lupin (Lupinus albus L.) cv. AMIGA. We retrieved the PacBio genome assembly scaffolded by Hi-C [25] and used the corresponding optical maps [26] to validate the assembly. This revealed 401 conflicts involving 27 scaffolds, 45 of which were located near gaps introduced by Hi-C and classified as true conflicts by manual inspection. The contiguity of the genome decreased by 50%, from 18 to 9 Mb (Table 3). The correctness of optical maps anchored scaffolds was manually verified by aligning the PacBio data to this assembly. Figure 3A shows PacBio reads properly aligned over the reconstruction obtained following the optical maps-based correction of a conflict site, supporting the final structure of the hybrid assembly and confirming the previous misplacement generated by Hi-C scaffolding (Figure 3B).
The same approach was then applied to three randomly selected genomes from the Vertebrate Genome Project: the Hawaiian crow (Corvus hawaiiensis), the South Island Takahe (Porphyrio hotchstetteri) and the American shad (Alosa sapidissima). All three assemblies are based on PacBio contigs anchored to optical maps before scaffolding with Hi-C data. The final genome assemblies were validated against the same optical maps by using the hybrid assembly procedure. We identified multiple conflicts between the optical maps and Hi-C scaffolds (352 for C. hawaiiensis, 182 for P. hotchstetteri and 825 for A. sapidissima) involving 43%, 42% and 82% of the scaffolds, respectively. Such conflicts are, therefore, widespread and can involve a large proportion of the chromosomes (Table 4). Among the identified conflicts, 5 (C. hawaiiensis), 11 (P. hotchstetteri) and 126 (A. sapidissima) were located near gaps introduced by Hi-C, but not due to Hi-C wrong gap-estimate and thus classified as Hi-C errors by manual inspection.
After optical maps conflict resolution, the contiguity of all three genomes decreased significantly (Table 5). The greatest impact was observed for A. sapidissima, where the scaffold N50 value decreased by order of magnitude from 38 to 3.6 Mb. However, following the integration of optical maps for scaffolding, the final hybrid assembly of all three genomes had a similar contiguity to the corresponding starting assembly in terms of scaffold N50 value. Moreover, the size of the gaps increased 10-fold (C. hawaiiensis and A. sapidissima) or threefold (P. hotchstetteri).

3. Discussion

Traditionally, the validation of de novo assemblies has relied on physical maps, such as those based on radiation hybrids and fluorescence in situ hybridization. More recently, innovative technologies such as optical mapping and electronic mapping have been developed to provide equivalent validation capability. We used optical maps after Hi-C scaffolding to assess the robustness of Hi-C reconstructions. We observed hundreds of incongruences in all the genome assemblies we analysed, ranging from 182 in P. hotchstetteri to 864 in H. lacustris, thus highlighting the discordance between the two reconstruction methods. Notably, a large fraction of conflicts was located near Hi-C junctions, suggesting possible misjoins reflecting the intrinsic properties of the Hi-C method. Confirming this hypothesis, we found that 83% of the putative misjoins in H. lacustris were also supported by ONT long reads, with similar results in the other genome assemblies. Furthermore, the correctness of optical map-based reconstruction was confirmed by aligning raw DNA molecules to the assembled maps.
As it was already reported in the literature, Hi-C is prone to generate contig misorientation and misplacement because chromosome structure is estimated statistically based on contact frequency. Several other factors also cause Hi-C errors. First, Hi-C is based on short-read sequencing that cannot uniquely map over genomic repeats, leading to low-quality alignments and the uneven distribution of genomic data. Second, restriction enzymes are used to generate the final NGS library, so performance depends on the frequency and distribution of restriction sites in the genome, potentially leading to the sparse mapping or complete omission of some contigs.
Interestingly, Hi-C errors were not restricted to low-complexity regions such as telomers or centromeres but were spread throughout most of the reconstructed chromosomes. For example, the manually identified 126 Hi-C errors in the A. sapidissima genome assembly were found in 21 out of 29 (72%) reconstructed chromosomes. Furthermore, in contrast to earlier reports suggesting that Hi-C errors are more prevalent among short contigs, we found no correlation between the error rate and contig length in the H. lacustris genome. The contig length with the highest frequency of errors was among the longest (600–650 kb).
The importance of optical maps as a validation technology does not only reflect the identification of scaffolding errors. Indeed, the hybrid scaffolding pipeline identifies incongruences with the NGS-based reconstructions in the first step, and the corrected sequences are anchored to the maps to generate more reliable scaffolds in the second step. Although the first step introduces “cuts” corresponding to the incongruences, integration of the maps as a scaffolding tool in the second step can restore the contiguity of the assembly. Accordingly, the N50 value of the final scaffolds may not differ considerably following the correction. This was observed in four of the five assemblies we analysed. The N50 value of the P. Hotchstetteri assembly was identical before and after optical maps integration. In A. sapidissima, which featured the greatest number of incongruences among the publicly available genome assemblies, the N50 value was only halved by hybrid scaffolding. The exception was H. lacustris, where the hybrid assembly significantly decreased the assembly N50 value, probably due to the high content of repeat sequences in this genome.
A second advantage provided by the optical maps hybrid scaffolding is the correct estimation of gap sizes. Optical map scaffolding is based on the measurement of physical distances between markers, whereas Hi-C cannot determine the correct gap size and introduces a user-determined fixed gap length. This was particularly evident in the H. lacustris assembly, where none of the gaps was correctly sized. In all the other genomes, the gap size increased up to 10-fold following the integration of optical maps. Another common limitation of NGS de novo genome assembly is tandem repeat collapsing [27], where near identical repeats are assembled as a single copy or the incorrect number of copies. This was evident in the recent telomere-to-telomere reconstruction of the human genome, where the only unresolved region was the tandem array of rRNA genes [8]. Optical maps are sequence-agnostic and can therefore provide accurate estimates for very long regions of tandem repeats [28], even those that defeat long-read sequencing.
In conclusion, in agreement with earlier studies, we demonstrated that Hi-C scaffolding could lead to contig misorientation and misidentified joints, but we suggest that these errors can be resolved by using optical maps as the final scaffolding step in order to improve the structural accuracy of genome assemblies. One possible limitation of optical mapping is its low resolution. With a density of one label every ~10 kb, only contigs longer than ~100 kb contain a sufficient amount of label sites for confident alignment [29]. A new technology from Nabsys for high-throughput electronic mapping, which is based on solid-state nanodetectors [30] and expected to provide higher resolution and accuracy than optical mapping, may overcome the limitations caused by low label density. Furthermore, current long-read technologies can easily generate de novo assemblies with contigs longer than 1 Mb [31,32,33].

4. Materials and Methods

4.1. Haematococcus lacustris Cultivation

We obtained H. lacustris strain K0084 from the Culture Collection of Algae at Goettingen University. Cells were seeded into flasks containing BG-11 medium and were grown photoautotrophically at 25 °C.

4.2. ONT Sequencing

Nuclei were isolated from 4.3 × 108 H. lacustris cells in MEB buffer [34] (Lutz, 2011), and the nuclear DNA was extracted using the Qiagen Genomic Tip-100 (Qiagen, Hilden, Germany). After DNA quantification and quality control as above, the nuclear DNA was fragmented to ~20 kb using a g-TUBE (Covaris, Brighton, UK) and treated with a short read eliminator (Circulomics, Pacific Biosciences, Menlo Park, CA, USA) to remove short fragments [35]. A 4-µg aliquot of DNA was end-repaired and dA-tailed using the Next End Repair/dA-tailing module (New England Biolabs, Ipswich, MA, USA) and ONT libraries were prepared using the ligation protocol (SQK-LSK109) according to the manufacturer’s instructions (ONT, Oxford, UK) but with longer incubation times [36]. Approximately 15 fmol of the library was loaded into a MinION flowcell (FLO-MIN106_R9.4.1), and loading was repeated on the same flowcell after nuclease flushing (NFL_9076_v109). The sequencing run lasted ~48 h.

4.3. Illumina Sequencing

Whole-genome Illumina DNA-Seq libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems, Wilmington, MA, USA) and a PCR-free protocol. Nuclear DNA was sheared using an M220 ultra-sonicator (Covaris), adjusting the treatment time to obtain ~350-bp fragments. The size of the resulting libraries was assessed by capillary electrophoresis on a Bioanalyzer High Sensitivity DNA chip. Libraries were quantified by qPCR using a standard curve and were sequenced on an Illumina NovaSeq 6000 device to generate 119 million 150 paired-end reads (36 Gb).

4.4. Optical Genome Mapping

UHMW DNA was extracted from isolated H. lacustris nuclei using agarose plugs (Bionano Genomics, San Diego, CA, USA). After DNA quality control as above, 300 ng of DNA was labelled using 10 U/µL Nt.BspQI or 20 U/µL Nb.BssSI nicking endonuclease (New England Biolabs, Ipswich, MA, USA) with the NLRS DNA labelling kit (Bionano Genomics). Maps were acquired from nicked and labelled DNA using a Syphyr instrument (Bionano Genomics) at the ETH functional Genomic Center of the University of Zurich.

4.5. Hi-C Data Generation

We fixed H. lacustris biomass in 1% fresh formaldehyde for 20 min and then quenched it with 1.25 mM glycine. Nuclei were isolated in NIBTM buffer [37] (and Hi-C libraries were prepared using the Proximo Hi-C Plant kit v1.5 (Phase Genomics, Seattle, WA, USA) and restriction enzyme Sau3AI. The integrity and size distribution of the Hi-C library was assessed using a 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA).

4.6. H. Lacustris De Novo Genome Assembly

ONT raw reads were assembled using Flye v2.5 with default parameters [38]. The draft contig assembly underwent base-level refinement of residual errors using a combination of long and short reads. Briefly, ONT reads were aligned on the ONT draft assembly using minimap2 v2.17 with the-x map-ont parameter [39]. Racon v1.4.3 [40] was used for the initial polishing of long reads, followed by a second round using medaka v1.0.3 (https://github.com/nanoporetech/medaka (accessed on 20 July 2020)) and two rounds of sequence refinement using short reads in pilon v1.23 [41]. Genome completeness was assessed with BUSCO v4.0.6 [42] using chlorophyta_odb10 as a reference database. The purging and scaffolding of the polished ONT assembly using Hi-C data was carried out by Phase Genomics using the Proximo Genome Scaffolding Platform. After the automatic scaffolding procedure, the results were manually revised to correct putative errors.

4.7. Optical Genome Maps: Assembly and Hybrid Scaffolding

Optical genome maps obtained with two nicking enzymes (BspQI and BssSI) were independently assembled using Bionano Solve v3.6 (https://bionanogenomics.com/support/ (accessed on 10 December 2020)). Double enzyme hybrid scaffolding was generated from the Hi-C-scaffolded genome using Bionano Solve v3.6 and the hybridScaffold_two_enzymes_HiC.xml configuration file. The hybrid assemblies were imported to the Bionano Access system, and conflicts were manually verified.

4.8. Source of Publicly Available Sequencing Data

The Lupinus albus genome map was downloaded from https://www.whitelupin.fr/ (accessed on 20 December 2021) (Hufnagel B. et al.), and the Hi-C-scaffolded genome was sourced from NCBI Bioproject PRJNA592024 (Xu W. et al.). VGP assembly data for C. hawaiiensis, P. hotchstetteri and A. sapidissima were downloaded from https://genomeark.github.io/vgp-all (accessed on 20 December 2021). The NGS and map assemblies were processed by hybrid scaffolding using Bionano Solve v3.6 after modifying the nicking enzyme to match the ones used for acquisition. The hybrid assemblies were imported into the Bionano Access system, and conflicts were manually verified.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12020320/s1, Table S1: Statistical analysis of the Haematococcus lacustris sequencing and mapping data; Table S2: Statistical analysis of the Haematococcus lacustris draft genome assembly data; Table S3: Number of contigs in Hi-C scaffolds grouped by contig length and relative percentage of erroneous joins.

Author Contributions

Conceptualization M.D.; Methodology L.M. and M.D.; Software L.V., T.M.; Investigation L.M., E.C., T.M., A.L. and M.O.; Formal analysis T.M. and M.O.; Validation L.M., L.V. and I.F.; Resources M.B. and M.C.; Data Curation A.L. and M.C.; Writing—Original Draft L.M.; Writing—Review and Editing M.R. and M.D.; Visualization E.C.; Supervision M.D.; Project administration M.D.; Funding acquisition M.B., M.R. and M.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the ERC Starting Grant SOLENALGAE (679814) to M.B., by the University of Verona Joint Project JPVR18RY8Y to M.R. and by Riba2017 (RBVR17WKNJ) to M.D. The founding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw sequencing and mapping data presented in this work are openly available at NCBI database (Bioproject accession number PRJNA904687).

Conflicts of Interest

Author E.C. is an employee of Genartis srl. Authors M.R. and M.D. are partners of Genartis srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Plants 12 00320 g001 550
Figure 1. Example of inconsistency between optical maps and Hi-C-derived scaffolds in the H. lacustris assembly. (A) Alignment to the NGS assembly scaffolded with Hi-C. Red and green arrows indicate the incongruence points in the BspQI and the BssSI maps, respectively. (B) Raw Bionano molecules aligned on the two maps involved in the conflict.
Figure 1. Example of inconsistency between optical maps and Hi-C-derived scaffolds in the H. lacustris assembly. (A) Alignment to the NGS assembly scaffolded with Hi-C. Red and green arrows indicate the incongruence points in the BspQI and the BssSI maps, respectively. (B) Raw Bionano molecules aligned on the two maps involved in the conflict.
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Plants 12 00320 g002 550
Figure 2. Integrative Genome Browser of two junction regions identified as potential errors by optical maps. ONT data mapped to the Hi-C-scaffolded H. lacustris genome show either (A) no reads spanning the contig junction or (B) reads spanning the junction but highlighting the incorrect gap size.
Figure 2. Integrative Genome Browser of two junction regions identified as potential errors by optical maps. ONT data mapped to the Hi-C-scaffolded H. lacustris genome show either (A) no reads spanning the contig junction or (B) reads spanning the junction but highlighting the incorrect gap size.
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Plants 12 00320 g003 550
Figure 3. Integrative Genome Browser, visualisation of long PacBio, reads aligned on an OM/Hi-C conflict identified on the L. albus genome assemblies. (A) PacBio reads aligned on the PacBio + Hi-C + OM (after anchoring) assembly support the OM-based reconstruction. (B) PacBio reads aligned on the PacBio + Hi-C assembly do not support the HiC-based reconstruction.
Figure 3. Integrative Genome Browser, visualisation of long PacBio, reads aligned on an OM/Hi-C conflict identified on the L. albus genome assemblies. (A) PacBio reads aligned on the PacBio + Hi-C + OM (after anchoring) assembly support the OM-based reconstruction. (B) PacBio reads aligned on the PacBio + Hi-C assembly do not support the HiC-based reconstruction.
Plants 12 00320 g003
Table
Table 1. Statistical data for the H. lacustris assembly (ONT assembly) scaffolded with Hi-C (ONT + Hi-C) and then corrected and anchored with optical maps (ONT + Hi-C + OM).
Table 1. Statistical data for the H. lacustris assembly (ONT assembly) scaffolded with Hi-C (ONT + Hi-C) and then corrected and anchored with optical maps (ONT + Hi-C + OM).
ONT AssemblyONT + Hi-CONT + Hi-C + OM
Total assembly length150,233,945150,327,121175,351,177
Total scaffold length (bp)-137,192,742162,448,219
Number of scaffolds-321507
Scaffold N50 (bp)-4,010,071669,654
Scaffold average length-4,287,273107,796
Longest scaffold (bp)-9,907,9704,400,370
Shortest scaffold (bp)-1,735,284210
Number of gaps-9301120
Gap size (bp)-93,00025,118,148
Contigs in scaffolds-9621696
Remaining contigs179984958
Remaining contig total length (bp)150,233,94513,134,37912,902,958
Remaining contig N50 (bp)230,87649,528248,743
Remaining contig average length (bp)83,51015,470222,465
Remaining contig maximum length (bp)987,270438,472925,569
Remaining contig minimum length (bp)50150180,956
Table
Table 2. Classification of junction errors based on manual evaluation of the alignment of ONT reads on the Hi-C-scaffolded H. lacustris genome assembly. The table shows the number and percentage of errors identified by alignment to optical maps and confirmed by manual inspection of ONT read mapping at the conflict site.
Table 2. Classification of junction errors based on manual evaluation of the alignment of ONT reads on the Hi-C-scaffolded H. lacustris genome assembly. The table shows the number and percentage of errors identified by alignment to optical maps and confirmed by manual inspection of ONT read mapping at the conflict site.
TypeNumberPercentage
Confirmed error50/6281%
Non-confirmed error12/6219%
Table
Table 3. Statistical data for the L. albus assembly generated with PacBio data (PacBio assembly [25]), the correction with optical maps (PacBio + Hi-C after OM conflict resolution) and the final hybrid scaffold integrating the PacBio assembly with Hi-C and optical maps scaffolding (PacBio + Hi-C + OM).
Table 3. Statistical data for the L. albus assembly generated with PacBio data (PacBio assembly [25]), the correction with optical maps (PacBio + Hi-C after OM conflict resolution) and the final hybrid scaffold integrating the PacBio assembly with Hi-C and optical maps scaffolding (PacBio + Hi-C + OM).
PacBio + Hi-CPacBio + Hi-C
After OM Conflict Resolution		PacBio + Hi-C + OM
Total assembly length (bp)558,896,430558,896,430566,549,843
Total scaffolds length (bp)500,601,628494,945,822506,701,445
Number of scaffolds67169124
Scaffold N50 (bp)18,661,2069,318,95911,119,386
Scaffold average length (bp)7,471,666.092,928,673.504,086,301.98
Longest scaffold (bp)25,248,48919,026,29822,043,127
Shortest scaffold (bp)11,44544394439
Number of gaps176417641823
Gap size (bp)1,532,8531,532,8539,186,266
Contigs in scaffolds183119331947
Remaining contigs151318121798
Remaining contig total length (bp)58,294,80263,950,60859,848,398
Table
Table 4. Classification of conflicts between optical maps and Hi-C scaffolds based on the manual evaluation of incongruent regions. The table shows the total number of conflicts identified by alignment to optical maps and the number of affected scaffolds compared to the total number of chromosomes.
Table 4. Classification of conflicts between optical maps and Hi-C scaffolds based on the manual evaluation of incongruent regions. The table shows the total number of conflicts identified by alignment to optical maps and the number of affected scaffolds compared to the total number of chromosomes.
Corvus hawaiiensisPorphyrio hotchstetteriAlosa sapidissima
Number of conflicts352182825
Number of scaffolds cut/Number of chromosomes21/4830/7124/29
Table
Table 5. Statistical data for the Corvus hawaiiensis, Porphyrio hotchstetteri and Alosa sapidissima scaffolded assemblies generated from PacBio data in the Vertebrate Genome Project (Hi-C), the assembly statistics after conflicts resolution (After OM conflict resolution) and the final hybrid scaffold integrating the PacBio assembly with Hi-C and optical maps scaffolding (OM.).
Table 5. Statistical data for the Corvus hawaiiensis, Porphyrio hotchstetteri and Alosa sapidissima scaffolded assemblies generated from PacBio data in the Vertebrate Genome Project (Hi-C), the assembly statistics after conflicts resolution (After OM conflict resolution) and the final hybrid scaffold integrating the PacBio assembly with Hi-C and optical maps scaffolding (OM.).
Corvus hawaiiensisPorphyrio hotchstetteriAlosa sapidissima
Hi-CAfter OM Conflict ResolutionOMHi-CAfter OM Conflict ResolutionOMHi-CAfter OM Conflict ResolutionOM
Total assembly length (bp)1,151,594,4811,151,593,2811,169,606,0521,270,322,6741,270,322,4341,280,192,390903,564,947903,564,347951,311,491
Total scaffold length (bp)1,089,055,265997,131,8281,136,185,4541,221,472,0231,167,038,0741,229,274,670898,428,452782,348,280924,915,787
Number of scaffolds4869657112310729378147
Scaffold N50 (bp)76,278,83235,694,30465,807,56271,566,19343,238,43971,566,19338,440,0663,604,00735,651,408
Scaffold average length (bp)22,688,651.3514,451,185.9117,479,776.2217,203,831.319,488,114.4211,488,548.3230,980,291.452,069,704.446,291,944.13
Longest scaffold (bp)123,451,405105,815,375120,194,775224,114,340105,985,913170,159,90256,504,57818,980,07062,312,241
Shortest scaffold (bp)86,66017,82617,82625,7682832283222,19212871287
Number of gaps294294384327327406163916411983
Gap size (bp)1,813,2271,813,22719,825,9985,316,2495,316,24915,186,2054,995,8124,995,81252,742,956
Contigs in scaffolds342363449398442506166819932108
Remaining contigs13947238610324217844522407
Remaining contig total length (bp)62,539,216154,461,45333,420,59848,850,651103,284,36050,917,7205,136,495121,216,06726,395,704
Remaining contig N50 (bp)16,938,3869,525,645551,25012,827,66016,562,10312,827,660233,9261,091,412130,353
remaining contig average length (bp)449,922.42327,248.8486,581.86474,278.17426,794.88286,054.61116,738.52232,214.6964,854.31
Longest remaining contig (bp)23,235,10033,421,9257,094,51719,484,41720,414,05919,484,417403,2926,672,8341,756,549
Shortest remaining contig (bp)229764164139125045042376659659
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Marcolungo, L.; Vincenzi, L.; Ballottari, M.; Cecchin, M.; Cosentino, E.; Mignani, T.; Limongi, A.; Ferraris, I.; Orlandi, M.; Rossato, M.; et al. Structural Refinement by Direct Mapping Reveals Assembly Inconsistencies near Hi-C Junctions. Plants 2023, 12, 320. https://doi.org/10.3390/plants12020320

AMA Style

Marcolungo L, Vincenzi L, Ballottari M, Cecchin M, Cosentino E, Mignani T, Limongi A, Ferraris I, Orlandi M, Rossato M, et al. Structural Refinement by Direct Mapping Reveals Assembly Inconsistencies near Hi-C Junctions. Plants. 2023; 12(2):320. https://doi.org/10.3390/plants12020320

Chicago/Turabian Style

Marcolungo, Luca, Leonardo Vincenzi, Matteo Ballottari, Michela Cecchin, Emanuela Cosentino, Thomas Mignani, Antonina Limongi, Irene Ferraris, Matteo Orlandi, Marzia Rossato, and et al. 2023. "Structural Refinement by Direct Mapping Reveals Assembly Inconsistencies near Hi-C Junctions" Plants 12, no. 2: 320. https://doi.org/10.3390/plants12020320

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