A beginner’s guide to assembling a draft genome and analyzing structural variants with long-read sequencing technologies

Kim, Jun; Kim, Chuna

doi:10.1016/j.xpro.2022.101506

Cited by 9 publications

(8 citation statements)

References 28 publications

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“…PacBio HiFi reads were filtered using BLASR (Chaisson and Tesler, 2012) to remove the PacBio 2kb sequence control. We employed a previously described approach for structural variant calling (Kim and Kim, 2022). We performed de novo assemblies using Flye 2.9-b1768 (Kolmogorov et al, 2020) with an estimated genome size of 135M and four polishing runs.…”

Section: Methodsmentioning

confidence: 99%

“…We performed de novo assemblies using Flye 2.9-b1768 (Kolmogorov et al, 2020) with an estimated genome size of 135M and four polishing runs. We assessed the quality of the assembled contigs by (1) visualization with Bandage 0.8.1 (Wick et al, 2015), (2) calculation of the cumulative coverage and the N50 value as described (Kim and Kim, 2022), and (3) testing for the completeness of the assembly using BUSCO v5.2.2 in genome mode against the brassicales_odb10 database (Manni et al, 2021). Next, contigs were used for scaffolding with Ragtag v2.1.0 (Alonge et al, 2022) with default parameters and the TAIR10 Arabidopsis reference genome (https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-55/fasta/arabidopsis_thaliana/).…”

Section: Long-read Sequencing (Pacbio)mentioning

confidence: 99%

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Targeted gene deletion withSpCas9 and multiple guide RNAs inArabidopsis thaliana: four are better than two

Ordon

Kiel

Becker

et al. 2023

Preprint

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Background. In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the fragment to be deleted. Experimental approaches for deletion induction have not been systematically evaluated. Results. We designed three pairs of guide RNAs for deletion of the Arabidopsis WRKY30 locus (~2.2 kb). We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal deletions. Conclusions. Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal deletions, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

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Section: Methodsmentioning

confidence: 99%

Section: Long-read Sequencing (Pacbio)mentioning

confidence: 99%

Targeted gene deletion withSpCas9 and multiple guide RNAs inArabidopsis thaliana: four are better than two

Ordon

Kiel

Becker

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…PacBio HiFi reads were filtered using BLASR [ 5 ] to remove the PacBio 2 kb sequence control. We employed a previously described approach for structural variant calling [ 26 ]. We performed de novo assemblies using Flye 2.9-b1768 [ 27 ] with an estimated genome size of 135 M and four polishing runs.…”

Section: Methodsmentioning

confidence: 99%

“…We performed de novo assemblies using Flye 2.9-b1768 [ 27 ] with an estimated genome size of 135 M and four polishing runs. We assessed the quality of the assembled contigs by (1) visualization with Bandage 0.8.1 [ 51 ], (2) calculation of the cumulative coverage and the N50 value as described [ 26 ], and (3) testing for the completeness of the assembly using BUSCO v5.2.2 in genome mode against the brassicales_odb10 database [ 34 ]. Next, contigs were used for scaffolding with Ragtag v2.1.0 [ 1 ] with default parameters and the TAIR10 Arabidopsis reference genome ( https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-55/fasta/arabidopsis_thaliana/ ).…”

Section: Methodsmentioning

confidence: 99%

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two

et al. 2023

View full text Add to dashboard Cite

Background In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. Results We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. Conclusions Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

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“…To obtain repeat-masked P. mannii and P. rapae genome versions, we followed Kim & Kim (2022) and first used RepeatMasker (v4.0.9; http://repeatmasker.org) with its internal Arthropoda repeat library (Dfam v3.0) to identify known repeats. De novo repeat libraries specific to the assembly of each Pieris species were then established using RepeatModeler (v2.0.1; http://repeatmasker.org).…”

Section: Dna Extraction Long-read Sequencing and Genome Assemblymentioning

confidence: 99%

Chromosome-level assemblies of thePieris manniibutterfly genome suggest Z-origin and rapid evolution of the W chromosome

Blattner

Ruffener

Berner

2023

Preprint

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The insect order Lepidoptera (butterflies and moth) represents the largest group of organisms with ZW/ZZ sex determination. While the origin of the Z chromosome predates the evolution of the Lepidoptera, the W chromosome is considered younger, but its origin is debated. To shed light on the origin of the lepidopteran W, we here produce chromosome-level genome assemblies for the butterfly Pieris mannii, and compare the sex chromosomes within and between P. mannii and its sister species P. rapae. Our analyses clearly indicate a common origin of the W chromosomes of the two Pieris species, and reveal similarity between the Z and W in chromosome sequence and structure. This supports the view that the W originates from Z-autosome fusion rather than from a redundant B chromosome. We further demonstrate the extremely rapid evolution of the W relative to the other chromosomes and argue that this may preclude reliable conclusions about the origin of the W based on comparisons among distantly related Lepidoptera. Finally, we find that sequence similarity between the Z and W chromosomes is greatest toward the chromosome ends, perhaps reflecting selection for the maintenance of recognition sites essential to chromosome segregation. Our study highlights the utility of long-read sequencing technology for illuminating chromosome evolution.

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A beginner’s guide to assembling a draft genome and analyzing structural variants with long-read sequencing technologies

Cited by 9 publications

References 28 publications

Targeted gene deletion withSpCas9 and multiple guide RNAs inArabidopsis thaliana: four are better than two

Targeted gene deletion withSpCas9 and multiple guide RNAs inArabidopsis thaliana: four are better than two

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two

Chromosome-level assemblies of thePieris manniibutterfly genome suggest Z-origin and rapid evolution of the W chromosome

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