Long‐fragment targeted capture for long‐read sequencing of plastomes

Bethune, Kévin; Mariac, Cédric; Couderc, Marie; Scarcelli, Nora; Santoni, Sylvain; Ardisson, Morgane; Martin, Jean; Montúfar, Rommel; Klein, Valentin; Sabot, François; Vigouroux, Yves; Couvreur, Thomas L. P.

doi:10.1002/aps3.1243

Cited by 27 publications

(22 citation statements)

References 32 publications

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“…However, improvements in the hybridization protocol are making the sequencing of captured fragments around 2 Kbp feasible, encouraging the use of long-read platforms in combination with target capture with the potential of increasing the completeness of the targeted region. For example, Bethune et al (2019) integrated target capture using a custom bait set, and sequencing using MinION ® (Oxford Nanopore Technologies), to produce long portions of the chloroplast; their method was successful for silicadried and fresh material of grasses and palms. Similarly, (Chen et al, 2018) designed a bait set from a frog DNA sample to recover targets from another two frog mitogenomes, then sequenced the targets using an Ion Torrent ™ Personal Genome Machine ™ .…”

Section: Sequencingmentioning

confidence: 99%

“…The lack of an established pipeline for long-read target capture is perhaps due to greater differences between datasets produced with these methods, in terms of coverage, fragment length, and intended purpose of the experiments. Nanopore sequencing is commonly used for assembling small genomes (e.g., Loman et al, 2015;Bethune et al, 2019) and while some workflows have been published as software packages, such as Nanopolish (based on Loman et al, 2015), the majority of studies using this sequencing technology apply a combination of bioinformatic toolbox commands to create project-specific workflows. Similarly, studies that have used PacBio sequencing in combination with target capture enrichment commonly create their individual customized workflows (e.g., Wang et al, 2015;Lagarde et al, 2017).…”

Section: Analytical Pipelinesmentioning

confidence: 99%

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A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

et al. 2020

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High-throughput DNA sequencing techniques enable time-and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.

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Section: Sequencingmentioning

confidence: 99%

Section: Analytical Pipelinesmentioning

confidence: 99%

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

et al. 2020

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“…Furthermore, the sensitivity of SV detection is improved by resolving variants in a haplotype-specific manner 38,42 . When long-read sequencing has been combined with specific target enrichment methods, using either a CRISPR/Cas9 system 43,44 or DNA probes 45,46 , it has been shown to yield accurate and contiguous assemblies. Such targeted approaches have enabled higher resolution genotyping of the HLA loci 47 and KIR regions 48,49 .…”

Section: Introductionmentioning

confidence: 99%

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

Rodriguez

Gibson

Parks

et al. 2020

Preprint

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An incomplete ascertainment of genetic variation within the highly polymorphic immunoglobulin heavy chain locus (IGH) has hindered our ability to define genetic factors that influence antibody and B cell mediated processes. To date, methods for locus-wide genotyping of all IGH variant types do not exist. Here, we combine targeted long-read sequencing with a novel bioinformatics tool, IGenotyper, to fully characterize genetic variation within IGH in a haplotype-specific manner. We apply this approach to eight human samples, including a haploid cell line and two mother-father-child trios, and demonstrate the ability to generate high-quality assemblies (>98% complete and >99% accurate), genotypes, and gene annotations, including 2 novel structural variants and 17 novel gene alleles. We show that multiplexing allows for scaling of the approach without impacting data quality, and that our genotype call sets are more accurate than short-read (>35% increase in true positives and >97% decrease in false-positives) and array/imputation-based datasets. This framework establishes a foundation for leveraging IG genomic data to study population-level variation in the antibody response.

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“…Only very few studies have applied Nanopore sequencing exclusively for plastome assembly. Bethune et al (2019) used Nanopore combined with target enrichment to sequence seven taxa from Poaceae and Palmaceae, 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.…”

Section: De Novo Assembly Based On Nanopore Data Alonementioning

confidence: 99%

“…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.…”

Section: De Novo Assembly Based On Nanopore Data Alonementioning

confidence: 99%

Can we use it? On the utility ofde novoand reference-based assembly of Nanopore data for plant plastome sequencing

Scheunert

Dorfner

Lingl

et al. 2019

Preprint

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The chloroplast genome harbors plenty of valuable information for phylogenetic research.Illumina short-read data is generally used for de novo assembly of whole plastomes. PacBio or Oxford Nanopore long reads are additionally employed in hybrid approaches to enable assembly across the highly similar inverted repeats of a chloroplast genome. Unlike for PacBio, plastome assemblies based solely on Nanopore reads are rarely found, due to their high error rate and non-random error profile. However, the actual quality decline connected to their use has never been quantified. Furthermore, no study has employed reference-based assembly using Nanopore reads, which is common with Illumina data. Using Leucanthemum Mill. as an example, we compared the sequence quality of seven plastome assemblies of the same species, using combinations of two sequencing platforms and three analysis pipelines.In addition, we assessed the factors which might influence Nanopore assembly quality during sequence generation and bioinformatic processing.The consensus sequence derived from de novo assembly of Nanopore data had a sequence identity of 99.59% compared to Illumina short-read de novo assembly. Most of the found errors comprise indels (81.5%), and a large majority of them is part of homopolymer regions.The quality of reference-based assembly is heavily dependent upon the choice of a closeenough reference. Using a reference with 0.83% sequence divergence from the studied species, mapping of Nanopore reads results in a consensus comparable to that from Nanopore de novo assembly, and of only slightly inferior quality compared to a referencebased assembly with Illumina data (0.49% and 0.26% divergence from Illumina de novo). For optimal assembly of Nanopore data, appropriate filtering of contaminants and chimeric sequences, as well as employing moderate read coverage, is essential.Based on these results, we conclude that Nanopore long reads are a suitable alternative to Illumina short reads in plastome phylogenomics. Only few errors remain in the finalized assembly, which can be easily masked in phylogenetic analyses without loss in analytical accuracy. The easily applicable and cost-effective technology might warrant more attention by researchers dealing with plant chloroplast genomes.

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Long‐fragment targeted capture for long‐read sequencing of plastomes

Cited by 27 publications

References 32 publications

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

Can we use it? On the utility ofde novoand reference-based assembly of Nanopore data for plant plastome sequencing

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