Comparison of long read methods for sequencing and assembly of a plant genome

Murigneux, Valentine; Kumar, Subash; Furtado, Agnelo; Bruxner, Timothy J. C.; Tian, Wei; Ye, Qianyu; Wei, Hanmin; Yang, Bicheng; Harliwong, Ivon; Anderson, Ellis; Mao, Qing; Drmanac, Radoje; Wang, Ou; Peters, Brock A.; Xu, Mengyang; Wu, Pei‐Ju; Topp, Bruce; Coin, Lachlan; Henry, Robert J.

doi:10.1101/2020.03.16.992933

Cited by 13 publications

(11 citation statements)

References 34 publications

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“…Although this benchmark gave essential information on the performance of these assemblers, eukaryote genomes represent a completely different challenge. Recently, a publication compared different long-read sequencing technologies to assemble a plant genome, Macadamia jansenii 31 . They included statistics for different assemblers and obtained, depending on the tool, oversized assemblies combined with heightened numbers of duplicated BUSCO features on an 80X PacBio dataset, while they did not observe such differences on a 30X Nanopore dataset.…”

Section: Discussionmentioning

confidence: 99%

Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms

Guiglielmoni

Houtain

Derzelle

et al. 2020

Preprint

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Third-generation sequencing, also called long-read sequencing, has revolutionized genome assembly: as PacBio and Nanopore technologies have become more accessible in technicity and in cost (with decreasing error rates and increasing read lengths), longread assemblers have flourished and are starting to deliver chromosome-level assemblies. However, an independent, comparative assessment of the performance of these programs on a common, real-life dataset is still lacking. To fill this gap, we tested the efficiency of long-read assemblers on the genome of the rotifer Adineta vaga, a non-model organism for which both PacBio and Nanopore reads were available. Although all the assemblers included in our benchmark aimed to produce a haploid genome assembly with collapsed haplotypes, we observed strikingly different behaviors of these assemblers on highly heterozygous regions: allelic regions that were most divergent were sometimes not merged, resulting in variable amounts of duplicated regions. We identified three strategies to alleviate this problem: setting a read-length threshold to filter out shorter reads; choosing an assembler less prone to retaining uncollapsed haplotypes; and post-processing the assembled set of contigs using a downstream tool to remove uncollapsed haplotypes. These three strategies are not mutually exclusive and, when combined, generate haploid assemblies with genome sizes, coverage distributions, and k-mer completeness matching expectations.

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Section: Discussionmentioning

confidence: 99%

Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms

Guiglielmoni

Houtain

Derzelle

et al. 2020

Preprint

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“…Accession numbers are as follows: BGI (SRR11191908), PacBio (SRR11191909), ONT PromethION (SRR11191910), ONT MinION (SRR11191911), and Illumina (SRR11191912). Assemblies and other supporting data are available from the GigaScience GigaDB repository [ 56 ].…”

Section: Data Availabilitymentioning

confidence: 99%

Comparison of long-read methods for sequencing and assembly of a plant genome

et al. 2020

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Background Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

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“…It demonstrates a possibility of combining SLR data with other data type, for instance, PacBio and ONT long reads, as well as other bioinformatics tools, for instance, single-nucleotide-polymorphism or structuralvariation callers. A typical association with long reads relates to the extension of scaffolds 48 and contigs 49,50 . It is necessary to take full advantages of different sequencing platforms and comprehensively utilize genomic information in short range (short reads, <1kb), medium range (long reads, 1k~10kb) and long range (ultra-long reads, SLR, Hi-C, BioNano, 10kb~Mb) with various error rates and balance their costs.…”

Section: Discussionmentioning

confidence: 99%

Haplotype-Resolved Assembly for Synthetic Long Reads Using a Trio-Binning Strategy

Xu¹,

Guo

et al. 2020

Preprint

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The accuracy and completeness of genome haplotyping are crucial for characterizing the relationship between human disease susceptibility and genetic variations, especially for the heterozygous variations. However, most of current variations are unphased genotypes, and the construction of long-range haplotypes remains challenging. We introduced a de novo haplotype-resolved assembly tool, HAST that exports two haplotypes of a diploid species for synthetic long reads with trio binning.It generates parental distinguishing k-mer libraries, partitions reads from the offspring according to the unique markers, and individually assembles them to resolve the haplotyping problem. Based on the stLFR co-barcoding data of an Asian as well as his parental massive parallel sequencing data, we utilized HAST to recover both haplotypes with a scaffold N50 of >11 Mb and an assembly accuracy of 99.99995% (Q63). The complete and accurate employment of long-range haplotyping information provided sub-chromosome level phase blocks (N50 ~13 Mb) with 99.6% precision and 94.1% recall on average. We suggest that the accurate and efficient approach accomplishes the regeneration of the haplotype chromosomes with trio binning, thus promoting the determination of haplotype phase, the heterosis of crossbreeding, and the formation of autopolyploid and allopolyploid.Page 3 haplotyping at its source. However, it is technically impractical for most species.Another strategy refers to the whole-chromosome isolation, for instance, chromosome sorting 6 , which is not applicable to large sample size due to its time-consuming and labor-intensive procedure. Haplotyping information can also be straightforwardly obtained from the sequencing of fragmented or whole DNA molecules. This approach requires long-range information to reconstruct the haplotypes from the biological or physical markers or overlapping relations of the segmented homologous chromosomes, including synthetic long reads (SLR, such as MGI stLFR co-barcoding reads and 10X genomics linked reads) 7-10 , single-molecule long reads (Pacific Biosciences (PacBio) or Oxford Nanopore (ONT) long reads) 11, 12 , Hi-C 13, 14 and optical mapping (Bionano) 15 . In practice the long-range information is employed to recover diploid or polyploid genomes with read-mapping-based, or de novo assemblybased methods.Currently, to attribute the successive genotypes to an arbitrary haplotype, sequencing reads are generally mapped to the reference genomes and then the alignment information recovers the heterozygous genotypes at each polymorphic site 16,17 .Nevertheless, the read-mapping-based methods are hampered by the lack of perfect reference sequence and the inability to detect large structural variations. Long reads 18, 19 or SLR 7,8,20 can extend the phase blocks to ~1Mb, but it is still complex to span regions of low heterozygous density or gaps in the reference. Assembly-based methods usually generate longer phase blocks across homozygous regions of the

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Comparison of long read methods for sequencing and assembly of a plant genome

Cited by 13 publications

References 34 publications

Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms

Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms

Comparison of long-read methods for sequencing and assembly of a plant genome

Haplotype-Resolved Assembly for Synthetic Long Reads Using a Trio-Binning Strategy

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